Very long chain ceramides interfere with C16-ceramide-induced channel formation: A plausible mechanism for regulating the initiation of intrinsic apoptosis

Mitochondria mediate both cell survival and death. The intrinsic apoptotic pathway is initiated by the permeabilization of the mitochondrial outer membrane to pro-apoptotic inter-membrane space (IMS) proteins. Many pathways cause the egress of IMS proteins. Of particular interest is the ability of ceramide to self-assemble into dynamic water-filled channels. The formation of ceramide channels is regulated extensively by Bcl-2 family proteins and dihydroceramide. Here, we show that the chain length of biologically active ceramides serves as an important regulatory factor. Ceramides are synthesized by a family of six mammalian ceramide synthases (CerS) each of which produces a subset of ceramides that differ in their fatty acyl chain length. Various ceramides permeabilize mitochondria differentially. Interestingly, the presence of very long chain ceramides reduces the potency of C₁₆-mediated mitochondrial permeabilization indicating that the intercalation of the lipids in the dynamic channel has a destabilizing effect, reminiscent of dihydroceramide inhibition of ceramide channel formation (Stiban et al., 2006). Moreover, mitochondria isolated from cells overexpressing the ceramide synthase responsible for the production of C₁₆-ceramide (CerS5) are permeabilized faster upon the exogenous addition of C₁₆-ceramide whereas they are resistant to permeabilization with added C₂₄-ceramide. On the other hand mitochondria isolated from CerS2-overexpressing cells show the opposite pattern, indicating that the product of CerS2 inhibits C₁₆-channel formation ex vivo and vice versa. This interplay between different ceramide metabolic enzymes and their products adds a new dimension to the complexity of mitochondrial-mediated apoptosis, and emphasizes its role as a key regulatory step that commits cells to life or death.     

Selective knockdown of ceramide synthases reveals complex interregulation of sphingolipid metabolism

Mammalian ceramide synthases 1 to 6 (CerS1-6) generate Cer in an acyl-CoA-dependent manner, and expression of individual CerS has been shown to enhance the synthesis of ceramides with particular acyl chain lengths. However, the contribution of each CerS to steady-state levels of specific Cer species has not been evaluated. We investigated the knockdown of individual CerS in the MCF-7 human breast adenocarcinoma cell line by using small-interfering RNA (siRNA). We found that siRNA-induced downregulation of each CerS resulted in counter-regulation of nontargeted CerS. Additionally, each CerS knockdown produced unique effects on the levels of multiple sphingolipid species. For example, downregulation of CerS2 decreased very long-chain Cer but increased levels of CerS4, CerS5, and CerS6 expression and upregulated long-chain and medium-long-chain sphingolipids. Conversely, CerS6 knockdown decreased C16:0-Cer but increased CerS5 expression and caused non-C16:0 sphingolipids to be upregulated. Knockdown of individual CerS failed to decrease total sphingolipids or upregulate sphingoid bases. Treatment with siRNAs targeting combined CerS, CerS2, CerS5, and CerS6, did not change overall Cer or sphingomyelin mass but caused upregulation of dihydroceramide and hexosyl-ceramide and promoted endoplasmic reticulum stress. These data suggest that sphingolipid metabolism is robustly regulated by both redundancy in CerS-mediated Cer synthesis and counter-regulation of CerS expression.     

Characterization of ceramide synthase 2: tissue distribution, substrate specificity, and inhibition by sphingosine 1-phosphate

Ceramide is an important lipid signaling molecule and a key intermediate in sphingolipid biosynthesis. Recent studies have implied a previously unappreciated role for the ceramide N-acyl chain length, inasmuch as ceramides containing specific fatty acids appear to play defined roles in cell physiology. The discovery of a family of mammalian ceramide synthases (CerS), each of which utilizes a restricted subset of acyl-CoAs for ceramide synthesis, strengthens this notion. We now report the characterization of mammalian CerS2. qPCR analysis reveals that CerS2 mRNA is found at the highest level of all CerS and has the broadest tissue distribution. CerS2 has a remarkable acyl-CoA specificity, showing no activity using C16:0-CoA and very low activity using C18:0, rather utilizing longer acyl-chain CoAs (C20-C26) for ceramide synthesis. There is a good correlation between CerS2 mRNA levels and levels of ceramide and sphingomyelin containing long acyl chains, at least in tissues where CerS2 mRNA is expressed at high levels. Interestingly, the activity of CerS2 can be regulated by another bioactive sphingolipid, sphingosine 1-phosphate (S1P), via interaction of S1P with two residues that are part of an S1P receptor-like motif found only in CerS2. These findings provide insight into the biochemical basis for the ceramide N-acyl chain composition of cells, and also reveal a novel and potentially important interplay between two bioactive sphingolipids that could be relevant to the regulation of sphingolipid metabolism and the opposing functions that these lipids play in signaling pathways.     

Ceramide synthase 4 and de novo production of ceramides with specific N-acyl chain lengths are involved in glucolipotoxicity-induced apoptosis of INS-1 β-cells

Pancreatic β-cell apoptosis induced by palmitate requires high glucose concentrations. Ceramides have been suggested to be important mediators of glucolipotoxicity-induced β-cell apoptosis. In INS-1 β-cells, 0.4 mM palmitate with 5 mM glucose increased the levels of dihydrosphingosine and dihydroceramides, two lipid intermediates in the de novo biosynthesis of ceramides, without inducing apoptosis. Increasing glucose concentrations to 30 mM amplified palmitate-induced accumulation of dihydrosphingosine and the formation of (dihydro)ceramides. Of note, glucolipotoxicity specifically induced the formation of C(18:0), C(22:0) and C(24:1) (dihydro)ceramide molecular species, which was associated with the up-regulation of CerS4 (ceramide synthase 4) levels. Fumonisin-B1, a ceramide synthase inhibitor, partially blocked apoptosis induced by glucolipotoxicity. In contrast, apoptosis was potentiated in the presence of D,L-threo-1-phenyl-2-palmitoylamino-3-morpholinopropan-1-ol, an inhibitor of glucosylceramide synthase. Moreover, overexpression of CerS4 amplified ceramide production and apoptosis induced by palmitate with 30 mM glucose, whereas down-regulation of CerS4 by siRNA (short interfering RNA) reduced apoptosis. CerS4 also potentiates ceramide accumulation and apoptosis induced by another saturated fatty acid: stearate. Collectively, our results suggest that glucolipotoxicity induces β-cell apoptosis through a dual mechanism involving de novo ceramide biosynthesis and the formation of ceramides with specific N-acyl chain lengths rather than an overall increase in ceramide content.     

Inactivation of Ceramide Synthase 6 in Mice Results in an Altered Sphingolipid Metabolism and Behavioral Abnormalities

The N-acyl chain length of ceramides is determined by the specificity of different ceramide synthases (CerS). The CerS family in mammals consists of six members with different substrate specificities and expression patterns. We have generated and characterized a mouse line harboring an enzymatically inactive ceramide synthase 6 (CerS6KO) gene and lacz reporter cDNA coding for β-galactosidase directed by the CerS6 promoter. These mice display a decrease in C16:0 containing sphingolipids. Relative to wild type tissues the amount of C16:0 containing sphingomyelin in kidney is ∼35%, whereas we find a reduction of C16:0 ceramide content in the small intestine to about 25%. The CerS6KO mice show behavioral abnormalities including a clasping abnormality of their hind limbs and a habituation deficit. LacZ reporter expression in the brain reveals CerS6 expression in hippocampus, cortex, and the Purkinje cell layer of the cerebellum. Using newly developed antibodies that specifically recognize the CerS6 protein we show that the endogenous CerS6 protein is N-glycosylated and expressed in several tissues of mice, mainly kidney, small and large intestine, and brain.     

Ceramide synthases 2, 5, and 6 confer distinct roles in radiation-induced apoptosis in HeLa cells

The role of ceramide neo-genesis in cellular stress response signaling is gaining increasing attention with recent progress in elucidating the novel roles and biochemical properties of the ceramide synthase (CerS) enzymes. Selective tissue and subcellular distribution of the six mammalian CerS isoforms, combined with distinct fatty acyl chain length substrate preferences, implicate differential functions of specific ceramide species in cellular signaling. We report here that ionizing radiation (IR) induces de novo synthesis of ceramide to influence HeLa cell apoptosis by specifically activating CerS isoforms 2, 5, and 6 that generate opposing anti- and pro-apoptotic ceramides in mitochondrial membranes. Overexpression of CerS2 resulted in partial protection from IR-induced apoptosis whereas overexpression of CerS5 increased apoptosis in HeLa cells. Knockdown studies determined that CerS2 is responsible for all observable IR-induced C_24:0 CerS activity, and while CerS5 and CerS6 each confer ～ 50% of the C_16:0 CerS baseline synthetic activity, both are required for IR-induced activity. Additionally, co-immunoprecipitation studies suggest that CerS2, 5, and 6 might exist as heterocomplexes in HeLa cells, providing further insight into the regulation of CerS proteins. These data add to the growing body of evidence demonstrating interplay among the CerS proteins in a stress stimulus-, cell type- and subcellular compartment-specific manner.     

Very long chain sphingolipids: Tissue expression, function and synthesis

Forecort membranes of all mammalian cells contain, in addition to phosphoglycerolipids and cholesterol, substantial amounts of sphingolipids. In most cells the acyl moieties of these sphingolipids are of long chain type (C16-24) and often saturated. However, epidermal keratinocytes and male germ cells largely express sphingolipids with very long chain-acyl moieties (C26-C36) during their differentiation and maturation. This expression seems to depend on CerS3, one of six ceramide synthases. However, the complex biosynthetic sequence for both epidermal and testicular sphingolipids has not been elucidated completely. Whereas it is established that omega-hydroxylated very long chain-sphingolipids are essential for proper skin barrier function, the role of polyunsaturated very long chain-sphingolipids (ceramides, sphingomyelins and glycosphingolipids) in differentiating spermatocytes/spermatids is just beginning to be revealed.     

Acyl chain specificity of ceramide synthases is determined within a region of 150 residues in the Tram-Lag-CLN8 (TLC) domain

In mammals, ceramides are synthesized by a family of six ceramide synthases (CerS), transmembrane proteins located in the endoplasmic reticulum, where each use fatty acyl-CoAs of defined chain length for ceramide synthesis. Little is known about the molecular features of the CerS that determine acyl-CoA selectivity. We now explore CerS structure-function relationships by constructing chimeric proteins combining sequences from CerS2, which uses C22-CoA for ceramide synthesis, and CerS5, which uses C16-CoA. CerS2 and -5 are 41% identical and 63% similar. Chimeras containing approximately half of CerS5 (from the N terminus) and half of CerS2 (from the C terminus) were catalytically inactive. However, the first 158 residues of CerS5 could be replaced with the equivalent region of CerS2 without affecting specificity of CerS5 toward C16-CoA; likewise, the putative sixth transmembrane domain (at the C terminus) of CerS5 could be replaced with the corresponding sequence of CerS2 without affecting CerS5 specificity. Remarkably, a chimeric CerS5/2 protein containing the first 158 residues and the last 83 residues of CerS2 displayed specificity toward C16-CoA, and a chimeric CerS2/5 protein containing the first 150 residues and the last 79 residues of CerS5 displayed specificity toward C22-CoA, demonstrating that a minimal region of 150 residues is sufficient for retaining CerS specificity.     

Developmentally regulated ceramide synthase 6 increases mitochondrial Ca2+ loading capacity and promotes apoptosis

Ceramides, which are membrane sphingolipids and key mediators of cell-stress responses, are generated by a family of (dihydro) ceramide synthases (Lass1-6/CerS1-6). Here, we report that brain development features significant increases in sphingomyelin, sphingosine, and most ceramide species. In contrast, C(16:0)-ceramide was gradually reduced and CerS6 was down-regulated in mitochondria, thereby implicating CerS6 as a primary ceramide synthase generating C(16:0)-ceramide. Investigations into the role of CerS6 in mitochondria revealed that ceramide synthase down-regulation is associated with dramatically decreased mitochondrial Ca(2+)-loading capacity, which could be rescued by addition of ceramide. Selective CerS6 complexing with the inner membrane component of the mitochondrial permeability transition pore was detected by immunoprecipitation. This suggests that CerS6-generated ceramide could prevent mitochondrial permeability transition pore opening, leading to increased Ca(2+) accumulation in the mitochondrial matrix. We examined the effect of high CerS6 expression on cell survival in primary oligodendrocyte (OL) precursor cells, which undergo apoptotic cell death during early postnatal brain development. Exposure of OLs to glutamate resulted in apoptosis that was prevented by inhibitors of de novo ceramide biosynthesis, myriocin and fumonisin B1. Knockdown of CerS6 with siRNA reduced glutamate-triggered OL apoptosis, whereas knockdown of CerS5 had no effect: the pro-apoptotic role of CerS6 was not stimulus-specific. Knockdown of CerS6 with siRNA improved cell survival in response to nerve growth factor-induced OL apoptosis. Also, blocking mitochondrial Ca(2+) uptake or decreasing Ca(2+)-dependent protease calpain activity with specific inhibitors prevented OL apoptosis. Finally, knocking down CerS6 decreased calpain activation. Thus, our data suggest a novel role for CerS6 in the regulation of both mitochondrial Ca(2+) homeostasis and calpain, which appears to be important in OL apoptosis during brain development.     

Inhibitors of specific ceramide synthases

Ceramide synthases (CerSs) are key enzymes in the biosynthesis of ceramides and display a group of at least six different isoenzymes (CerS1-6). Ceramides itself are bioactive molecules. Ceramides with different N-acyl side chains (C_14:0-Cer – C_26:0-Cer) possess distinct roles in cell signaling. Therefore, the selective inhibition of specific CerSs which are responsible for the formation of a specific ceramide holds promise for a number of new clinical treatment strategies, e.g., cancer. Here, we identified four of hitherto unknown functional inhibitors of CerSs derived from the FTY720 (Fingolimod) lead structure and showed their inhibitory effectiveness by two in vitro CerS activity assays. Additionally, we tested the substances in two cell lines (HCT-116 and HeLa) with different ceramide patterns. In summary, the in vitro activity assays revealed out that ST1058 and ST1074 preferentially inhibit CerS2 and CerS4, while ST1072 inhibits most potently CerS4 and CerS6. Importantly, ST1060 inhibits predominately CerS2. First structure–activity relationships and the potential biological impact of these compounds are discussed.     

Ablation of ceramide synthase 2 exacerbates dextran sodium sulphate‐induced colitis in mice due to increased intestinal permeability

Ceramides mediate crucial cellular processes including cell death and inflammation and have recently been implicated in inflammatory bowel disease. Ceramides consist of a sphingoid long‐chain base to which fatty acids of various length can be attached. We now investigate the effect of alerting the ceramide acyl chain length on a mouse model of colitis. Ceramide synthase (CerS) 2 null mice, which lack very‐long acyl chain ceramides with concomitant increase of long chain bases and C16‐ceramides, were more susceptible to dextran sodium sulphate‐induced colitis, and their survival rate was markedly decreased compared with that of wild‐type littermates. Using mixed bone‐marrow chimeric mice, we showed that the host environment is primarily responsible for intestinal barrier dysfunction and increased intestinal permeability. In the colon of CerS2 null mice, the expression of junctional adhesion molecule‐A was markedly decreased and the phosphorylation of myosin light chain 2 was increased. In vitro experiments using Caco‐2 cells also confirmed an important role of CerS2 in maintaining epithelial barrier function; CerS2‐knockdown via CRISPR‐Cas9 technology impaired barrier function. In vivo myriocin administration, which normalized long‐chain bases and C16‐ceramides of the colon of CerS2 null mice, increased intestinal permeability as measured by serum FITC‐dextran levels, indicating that altered SLs including deficiency of very‐long‐chain ceramides are critical for epithelial barrier function. In conclusion, deficiency of CerS2 influences intestinal barrier function and the severity of experimental colitis and may represent a potential mechanism for inflammatory bowel disease pathogenesis.     

Alteration of ceramide synthase 6/C16-ceramide induces activating transcription factor 6-mediated endoplasmic reticulum (ER) stress and apoptosis via perturbation of cellular Ca2+ and ER/Golgi membrane network

Mechanisms that regulate endoplasmic reticulum (ER) stress-induced apoptosis in cancer cells remain enigmatic. Recent data suggest that ceramide synthase1-6 (CerS1-6)-generated ceramides, containing different fatty acid chain lengths, might exhibit distinct and opposing functions, such as apoptosis versus survival in a context-dependent manner. Here, we investigated the mechanisms involved in the activation of one of the major ER stress response proteins, ATF-6, and subsequent apoptosis by alterations of CerS6/C(16)-ceramide. Induction of wild type (WT), but not the catalytically inactive mutant CerS6, increased tumor growth in SCID mice, whereas siRNA-mediated knockdown of CerS6 induced ATF-6 activation and apoptosis in multiple human cancer cells. Down-regulation of CerS6/C(16)-ceramide, and not its further metabolism to glucosylceramide or sphingomyelin, activated ATF-6 upon treatment with ER stress inducers tunicamycin or SAHA (suberoylanilide hydroxamic acid). Induction of WT-CerS6 expression, but not its mutant, or ectopic expression of the dominant-negative mutant form of ATF-6 protected cells from apoptosis in response to CerS6 knockdown and tunicamycin or SAHA treatment. Mechanistically, ATF-6 activation was regulated by a concerted two-step process involving the release of Ca(2+) from the ER stores ([Ca(2+)](ER)), which resulted in the fragmentation of Golgi membranes in response to CerS6/C(16)-ceramide alteration. This resulted in the accumulation of pro-ATF-6 in the disrupted ER/Golgi membrane network, where pro-ATF6 is activated. Accordingly, ectopic expression of a Ca(2+) chelator calbindin prevented the Golgi fragmentation, ATF-6 activation, and apoptosis in response to CerS6/C(16)-ceramide down-regulation. Overall, these data suggest a novel mechanism of how CerS6/C(16)-ceramide alteration activates ATF6 and induces ER-stress-mediated apoptosis in squamous cell carcinomas.     

Increasing ceramides sensitizes genistein-induced melanoma cell apoptosis and growth inhibition

The aim of the current study is to investigate the effect of ceramides on genistein-induced anti-melanoma effects in vitro. We found that exogenously added cell-permeable short-chain ceramides (C6) dramatically enhanced genistein-induced growth inhibition and apoptosis in cultured melanoma cells. Genistein treatment only induced a moderate intracellular ceramides accumulation in B16 melanoma cells. Two different agents including 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a ceramide glucosylation inhibitor, and the sphingosine kinase-1 (SphK1) inhibitor II (SKI-II), a sphingosine (ceramides precursor) phosphorylation inhibitor, both facilitated genistein-induced ceramides accumulation and melanoma cell apoptosis. Co-administration of ceramide (C6) and genistein induced a significant Akt inhibition and c-jun-NH(2)-kinase (JNK) activation, caspase-3 cleavage and cytochrome c release. Caspase-3 inhibitor z-DVED-fmk, JNK inhibitor SP 600125, or to restore Akt activation by introducing a constitutively active form of Akt (CA-Akt) diminished ceramide (C6) and genistein co-administration-induced in vitro anti-melanoma effect. Our study suggests that increasing cellular level of ceramides may sensitize genistein-induced anti-melanoma effects.     

Apoptosis occurs via the ceramide recycling pathway in human HaCaT keratinocytes

Keratinocytes contain abundant ceramides compared to other cells. However, studies on these cells have mainly focused on the barrier function of ceramide, while their other roles, such as those in apoptosis or cell cycle arrest, have not been well addressed. In this study, we investigated the apoptosis-inducing effect of exogenously added cell-permeable ceramides in HaCaT keratinocytes. We found that N-hexanoyl sphingosine (C6-ceramide) induced apoptosis efficiently through the accumulation of long chain ceramides. On the other hand, N-acetyl sphingosine (C2-ceramide) induced neither apoptosis nor accumulation of long chain ceramides. We also found that exogenously added C6-ceramide was hydrolyzed to sphingosine and then reacylated in long chain ceramides (ceramide recycling pathway), but that C2-ceramide was not hydrolyzed and thus not recycled. We propose that this is the basis for the chain length-specific heterogeneity observed in ceramide-induced apoptosis in these cells. These results also imply that keratinocytes utilize exogenous sphingolipids or ceramides to coordinate their own ceramide compositions.     

Ceramide synthases as potential targets for therapeutic intervention in human diseases

Ceramide is located at a key hub in the sphingolipid metabolic pathway and also acts as an important cellular signaling molecule. Ceramide contains one acyl chain which is attached to a sphingoid long chain base via an amide bond, with the acyl chain varying in length and degree of saturation. The identification of a family of six mammalian ceramide synthases (CerS) that synthesize ceramide with distinct acyl chains, has led to significant advances in our understanding of ceramide biology, including further delineation of the role of ceramide in various pathophysiologies in both mice and humans. Since ceramides, and the complex sphingolipids generated from ceramide, are implicated in disease, the CerS might potentially be novel targets for therapeutic intervention in the diseases in which the ceramide acyl chain length is altered. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

Ceramides and ceramide synthases in cancer: Focus on apoptosis and autophagy

Different studies corroborate a role for ceramide synthases and their downstream products, ceramides, in modulation of apoptosis and autophagy in the context of cancer. These mechanisms of regulation, however, appear to be context dependent in terms of ceramides’ fatty acid chain length, subcellular localization, and the presence or absence of their downstream targets. Our current understanding of the role of ceramide synthases and ceramides in regulation of apoptosis and autophagy could be harnessed to pioneer the development of new treatments to activate or inhibit a single type of ceramide synthase, thereby regulating the apoptosis induction or cross talk of apoptosis and autophagy in cancer cells. Moreover, the apoptotic function of ceramide suggests that ceramide analogues can pave the way for the development of novel cancer treatments. Therefore, in the current review paper we discuss the impact of ceramide synthases and ceramides in regulation of apoptosis and autophagy in context of different types of cancers. We also briefly introduce the latest information on ceramide synthase inhibitors, their application in diseases including cancer therapy, and discuss approaches for drug discovery in the field of ceramide synthase inhibitors. We finally discussed strategies for developing strategies to use lipids and ceramides analysis in biological fluids for developing early biomarkers for cancer.     

Modulation of ceramide synthase activity via dimerization

Ceramide, the backbone of all sphingolipids, is synthesized by a family of ceramide synthases (CerS) that each use acyl-CoAs of defined chain length for N-acylation of the sphingoid long chain base. CerS mRNA expression and enzymatic activity do not always correlate with the sphingolipid acyl chain composition of a particular tissue, suggesting post-translational mechanism(s) of regulation of CerS activity. We now demonstrate that CerS activity can be modulated by dimer formation. Under suitable conditions, high M(r) CerS complexes can be detected by Western blotting, and various CerS co-immunoprecipitate. CerS5 activity is inhibited in a dominant-negative fashion by co-expression with catalytically inactive CerS5, and CerS2 activity is enhanced by co-expression with a catalytically active form of CerS5 or CerS6. In a constitutive heterodimer comprising CerS5 and CerS2, the activity of CerS2 depends on the catalytic activity of CerS5. Finally, CerS dimers are formed upon rapid stimulation of ceramide synthesis by curcumin. Together, these data demonstrate that ceramide synthesis can be regulated by the formation of CerS dimers and suggest a novel way to generate the acyl chain composition of ceramide (and downstream sphingolipids), which may depend on the interaction of CerS with each other.     

Altering the sphingolipid acyl chain composition prevents LPS/GLN-mediated hepatic failure in mice by disrupting TNFR1 internalization

The involvement of ceramide in death receptor-mediated apoptosis has been widely examined with most studies focusing on the role of ceramide generated from sphingomyelin hydrolysis. We now analyze the effect of the ceramide acyl chain length by studying tumor necrosis factor α receptor-1 (TNFR1)-mediated apoptosis in a ceramide synthase 2 (CerS2) null mouse, which cannot synthesize very-long acyl chain ceramides. CerS2 null mice were resistant to lipopolysaccharide/galactosamine-mediated fulminant hepatic failure even though TNFα secretion from macrophages was unaffected. Cultured hepatocytes were also insensitive to TNFα-mediated apoptosis. In addition, in both liver and in hepatocytes, caspase activities were not elevated, consistent with inhibition of TNFR1 pro-apoptotic signaling. In contrast, Fas receptor activation resulted in the death of CerS2 null mice. Caspase activation was blocked because of the inability of CerS2 null mice to internalize the TNFR1; whereas Fc-TNFα was internalized to a perinuclear region in hepatocytes from wild-type mice, no internalization was detected in CerS2 null mice. Our results indicate that altering the acyl chain composition of sphingolipids inhibits TNFR1 internalization and inhibits selective pro-apoptotic downstream signaling for apoptosis.