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Dihydroceramide is a lipid molecule generated via the action of (dihydro)ceramide synthases (CerSs), which use two substrates, namely sphinganine and fatty acyl-CoAs. Sphinganine is generated via the sequential activity of two integral membrane proteins located in the endoplasmic reticulum. Less is known about the source of the fatty acyl-CoAs, although a number of cytosolic proteins in the pathways of acyl-CoA generation modulate ceramide synthesis via direct or indirect interaction with the CerSs. In this study, we demonstrate, by proteomic analysis of immunoprecipitated proteins, that fatty acid transporter protein 2 (FATP2) (also known as very long-chain acyl-CoA synthetase) directly interacts with CerS2 in mouse liver. Studies in cultured cells demonstrated that other members of the FATP family can also interact with CerS2, with the interaction dependent on both proteins being catalytically active. In addition, transfection of cells with FATP1, FATP2, or FATP4 increased ceramide levels although only FATP2 and 4 increased dihydroceramide levels, consistent with their known intracellular locations. Finally, we show that lipofermata, an FATP2 inhibitor which is believed to directly impact tumor cell growth via modulation of FATP2, decreased de novo dihydroceramide synthesis, suggesting that some of the proposed therapeutic effects of lipofermata may be mediated via (dihydro)ceramide rather than directly via acyl-CoA generation. In summary, our study reinforces the idea that manipulating the pathway of fatty acyl-CoA generation will impact a wide variety of down-stream lipids, not least the sphingolipids, which utilize two acyl-CoA moieties in the initial steps of their synthesis.

Oncogenic multidrug resistance (MDR) is commonly intrinsic to renal cancer based on the physiological expression of detoxification transporters, particularly ABCB1, thus hampering chemotherapy. ABCB1 activity is directly dependent on its lipid microenvironment, localizing to cholesterol- and sphingomyelin-rich domains. As ceramides are the sole source for sphingomyelins, we hypothesized that ceramide synthase (CerS)-derived ceramides regulate ABCB1 activity. Using data from RNA-seq databases, we found patient kidney tumors exhibited increased CerS2 mRNA, which was inversely correlated with CerS6 mRNA in ABCB1+ clear cell carcinomas. Endogenous elevated CerS2 and lower CerS5/6 mRNA and protein resulted in disproportionately higher CerS2 to CerS5/6 activities (2-fold) in chemoresistant ABCB1high (A498, Caki-1) compared to chemosensitive ABCB1low (ACHN, HPCT) cells. In addition, lipidomics analyses by HPLC-MS/MS showed bias towards CerS2-associated C20:0/C20:1-ceramides compared to CerS5/6-associated C14:0/C16:0-ceramides (2:1). Sphingomyelins were similarly altered. We demonstrated that chemoresistance to doxorubicin in ABCB1high cells was partially reversed by inhibitors of de novo ceramide synthesis (L-cycloserine) and CerS (fumonisin B1) in cell viability assays. Additionally, downregulation of CerS2/6, but not CerS5, attenuated ABCB1 mRNA, protein, plasma membrane localization, rhodamine 123+ efflux transport activity, and doxorubicin resistance. Similar findings were observed with catalytically-inactive CerS6-H212A. Furthermore, CerS6-targeting siRNA shifted ceramide and sphingomyelin composition to ultra long-chain species (C22-C26). Inhibitors of ER-associated degradation (ERAD) (eeyarestatin I) and the proteasome (MG132, bortezomib) prevented ABCB1 loss induced by CerS2/6 downregulation. We conclude that a critical balance in ceramide/sphingomyelin species is prerequisite to ABCB1 expression and functionalization, which could be targeted to reverse MDR in renal cancers. Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.

Ceramide is a lipid molecule that regulates diverse physiological and pathological reactions in part through inverting the topology of certain transmembrane proteins. This topological inversion is achieved through regulated alternative translocation (RAT), which reverses the direction by which membrane proteins are translocated across the endoplasmic reticulum during translation. However, owing to technical challenges in studying protein–ceramide interaction, it remains unclear how ceramide levels are sensed in cells to trigger RAT. Here, we report the synthesis of pac-C7-Cer, a photoactivatable and clickable short-chain ceramide analog that can be used as a probe to study protein–ceramide interactions. We demonstrate that translocating chain-associated membrane protein 2 (TRAM2), a protein known to control RAT of transmembrane 4 L6 subfamily member 20, and TRAM1, a homolog of TRAM2, interacted with molecules derived from pac-C7-Cer. This interaction was competed by naturally existing long-chain ceramide molecules. We showed that binding of ceramide and its analogs to TRAM2 correlated with their ability to induce RAT of transmembrane 4 L6 subfamily member 20. In addition to probing ceramide–TRAM interactions, we provide evidence that pac-C7-cer could be used for proteome-wide identification of ceramide-binding proteins. Our study provides mechanistic insights into RAT by identifying TRAMs as potential ceramide-binding proteins and establishes pac-C7-Cer as a valuable tool for future study of ceramide–protein interactions.

The acid sphingomyelinase/ceramide system has been shown to be important for cellular infection with at least some viruses, for instance, rhinovirus or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Functional inhibition of the acid sphingomyelinase using tricyclic antidepressants prevented infection of epithelial cells, for instance with SARS-CoV-2. The structure of ambroxol, that is, trans-4-[(2,4-dibromanilin-6-yl)methyamino]-cyclohexanol, a mucolytic drug applied by inhalation, suggests that the drug might inhibit the acid sphingomyelinase and thereby infection with SARS-CoV-2. To test this, we used vesicular stomatitis virus pseudoviral particles presenting SARS-CoV-2 spike protein on their surface (pp-VSV-SARS-CoV-2 spike), a bona fide system for mimicking SARS-CoV-2 entry into cells. Viral uptake and formation of ceramide localization were determined by fluorescence microscopy, activity of the acid sphingomyelinase by consumption of [¹⁴C]sphingomyelin and ceramide was quantified by a kinase method. We found that entry of pp-VSV-SARS-CoV-2 spike required activation of acid sphingomyelinase and release of ceramide, events that were all prevented by pretreatment with ambroxol. We also obtained nasal epithelial cells from human volunteers prior to and after inhalation of ambroxol. Inhalation of ambroxol reduced acid sphingomyelinase activity in nasal epithelial cells and prevented pp-VSV-SARS-CoV-2 spike-induced acid sphingomyelinase activation, ceramide release, and entry of pp-VSV-SARS-CoV-2 spike ex vivo. The addition of purified acid sphingomyelinase or C16 ceramide restored entry of pp-VSV-SARS-CoV-2 spike into ambroxol-treated epithelial cells. We propose that ambroxol might be suitable for clinical studies to prevent coronavirus disease 2019. CA - Gulbins

Diacylglycerol (DG) is a well-established lipid second messenger. Sphingomyelin synthase (SMS)-related protein (SMSr) produces DG and ceramide phosphoethanolamine (CPE) by the transfer of phosphoethanolamine from phosphatidylethanolamine (PE) to ceramide. We previously reported that human SMSr overexpressed in COS-7 cells significantly increased DG levels, particularly saturated and/or monounsaturated fatty acid–containing DG molecular species, and provided DG to DG kinase (DGK) δ, which regulates various pathophysiological events, including epidermal growth factor–dependent cell proliferation, type 2 diabetes, and obsessive–compulsive disorder. However, mammalian SMSr puzzlingly produces only trace amounts of CPE/DG. To clarify this discrepancy, we highly purified SMSr and examined its activities other than CPE synthase. Intriguingly, purified SMSr showed a DG-generating activity via hydrolysis of PE, phosphatidic acid (PA), phosphatidylinositol (PI), and phosphatidylcholine (PC) in the absence of ceramide. DG generation through the PA phosphatase (PAP) activity of SMSr was approximately 300-fold higher than that with PE and ceramide. SMSr hydrolyzed PI ten times stronger than PI(4,5)bisphosphate (PI(4,5)P2). The PAP and PC-phospholipase C (PLC) activities of SMSr were inhibited by propranolol, a PAP inhibitor, and by D609, an SMS/PC-PLC inhibitor. Moreover, SMSr showed substrate selectivity for saturated and/or monounsaturated fatty acid–containing PA molecular species, but not arachidonic-acid-containing PA, which is exclusively generated in the PI(4,5)P2 cycle. We confirmed that SMSr expressed in COS-7 cells showed PAP and PI-PLC activities. Taken together, our study indicated that SMSr possesses previously unrecognized enzyme activities, PAP and PI/PE/PC-PLC, and constitutes a novel DG/PA signaling pathway together with DGKδ, which is independent of the PI(4,5)P2 cycle.

The lipid composition of HIV-1 virions is enriched in sphingomyelin (SM), but the roles that SM or other sphingolipids (SLs) might play in the HIV-1 replication pathway have not been elucidated. In human cells, SL levels are regulated by ceramide synthase (CerS) enzymes that produce ceramides, which can be converted to SMs, hexosylceramides, and other SLs. In many cell types, CerS2, which catalyzes the synthesis of very long chain ceramides, is the major CerS. We have examined how CerS2 deficiency affects the assembly and infectivity of HIV-1. As expected, we observed that very long chain ceramide, hexosylceramide, and SM were reduced in CerS2 knockout cells. CerS2 deficiency did not affect HIV-1 assembly or the incorporation of the HIV-1 envelope (Env) protein into virus particles, but it reduced the infectivites of viruses produced in the CerS2-deficient cells. The reduced viral infection levels were dependent on HIV-1 Env, since HIV-1 particles that were pseudotyped with the vesicular stomatitis virus glycoprotein did not exhibit reductions in infectivity. Moreover, cell–cell fusion assays demonstrated that the functional defect of HIV-1 Env in CerS2-deficient cells was independent of other viral proteins. Overall, our results indicate that the altered lipid composition of CerS2-deficient cells specifically inhibit the HIV-1 Env receptor binding and/or fusion processes.

LPS triggers inflammatory responses; however, the negative regulation of LPS responses in vivo remains poorly understood. CD300f is an inhibitory receptor among the CD300 family of paired activating and inhibitory receptors. We have previously identified ceramide as a ligand for CD300f and shown that the binding of ceramide to CD300f inhibits IgE-mediated mast cell activation and allergic responses in mouse models. Here we identify the critical role of CD300f in inhibiting LPS-induced skin inflammation. CD300f deficiency remarkably enhanced LPS-induced skin edema and neutrophil recruitment in mice. Higher levels of factors that increase vascular permeability and of factors that induce neutrophil recruitment were detected in LPS-injected skin pouch exudates of CD300f-/- mice as compared with wild-type mice. CD300f was highly expressed in mast cells and recruited neutrophils, but not in macrophages, among skin myeloid cells. CD300f deficiency failed to influence the intrinsic migratory ability of neutrophils. Ceramide-CD300f binding suppressed the release of chemical mediators from mast cells and from neutrophils in response to LPS. Adoptive transfer experiments indicated that mast cells mediated enhanced edema in LPS-stimulated skin of CD300f-/- mice, whereas mast cells together with recruited neutrophils mediated robust neutrophil accumulation. Importantly, administering a ceramide antibody or ceramide-containing vesicles enhanced or suppressed LPS-induced skin inflammation of wild-type mice, respectively. Thus, ceramide-CD300f binding inhibits LPS-induced skin inflammation, implicating CD300f as a negative regulator of Toll-like receptor 4 (TLR4) signaling in vivo.

Sphingolipids compose a lipid family critical for membrane structure as well as intra- and intercellular signaling. De novo sphingolipid biosynthesis is initiated by the enzyme serine palmitoyltransferase (SPT), which resides in the endoplasmic reticulum (ER) membrane. In both yeast and mammalian species, SPT activity is homeostatically regulated through small ER membrane proteins, the Orms in yeast and the ORMDLs in mammalian cells. These proteins form stable complexes with SPT. In yeast, the homeostatic regulation of SPT relies, at least in part, on phosphorylation of the Orms. However, this does not appear to be the case for the mammalian ORMDLs. Here, we accomplished a cell-free reconstitution of the sphingolipid regulation of the ORMDL–SPT complex to probe the underlying regulatory mechanism. Sphingolipid and ORMDL-dependent regulation of SPT was demonstrated in isolated membranes, essentially free of cytosol. This suggests that this regulation does not require soluble cytosolic proteins or small molecules such as ATP. We found that this system is particularly responsive to the pro-apoptotic sphingolipid ceramide and that this response is strictly stereospecific, indicating that ceramide regulates the ORMDL–SPT complex via a specific binding interaction. Yeast membranes harboring the Orm–SPT system also directly responded to sphingolipid, suggesting that yeast cells have, in addition to Orm phosphorylation, an additional Orm-dependent SPT regulatory mechanism. Our results indicate that ORMDL/Orm-mediated regulation of SPT involves a direct interaction of sphingolipid with the membrane-bound components of the SPT-regulatory apparatus.

Creation of an intact skin water barrier, a prerequisite for life on dry land, requires the lipoxygenase-catalyzed oxidation of the essential fatty acid linoleate, which is esterified to the ω-hydroxyl of an epidermis-specific ceramide. Oxidation of the linoleate moiety by lipoxygenases is proposed to facilitate enzymatic cleavage of the ester bond, releasing free ω-hydroxyceramide for covalent binding to protein, thus forming the corneocyte lipid envelope, a key component of the epidermal barrier. Herein, we report the transformations of esterified linoleate proceed beyond the initial steps of oxidation and epoxyalcohol synthesis catalyzed by the consecutive actions of 12R-LOX and epidermal LOX3. The major end product in human and porcine epidermis is a trihydroxy derivative, formed with a specificity that implicates participation of an epoxide hydrolase in converting epoxyalcohol to triol. Of the 16 possible triols arising from hydrolysis of 9,10-epoxy-13-hydroxy-octadecenoates, using LC-MS and chiral analyses, we identify and quantify specifically 9R,10S,13R-trihydroxy-11E-octadecenoate as the single major triol esterified in porcine epidermis and the same isomer with lesser amounts of its 10R diastereomer in human epidermis. The 9R,10S,13R-triol is formed by SN2 hydrolysis of the 9R,10R-epoxy-13R-hydroxy-octadecenoate product of the LOX enzymes, a reaction specificity characteristic of epoxide hydrolase. The high polarity of triol over the primary linoleate products enhances the concept that the oxidations disrupt corneocyte membrane lipids, promoting release of free ω-hydroxyceramide for covalent binding to protein and sealing of the waterproof barrier.

The ceramide nanoliposome (CNL) has shown promise in being able to treat a variety of primary tumors. However, its potential for treating metastatic cancer remains unknown. In this study, we demonstrate that CNL increases anoikis while preventing cancer cell extravasation under both static and physiological fluid flow conditions. Mechanistically, CNL limits metastases by decreasing CD44 protein levels in human breast and pancreatic cancer cells via lysosomal degradation of CD44, independent of palmitoylation or proteasome targeting. siRNA down-regulation of CD44 mimics CNL-induced anoikis and diminished extravasation of cancer cells. Taken together, our data indicate that ceramide limits CD44-dependent cancer cell migration, suggesting that CNL could be used to prevent and treat solid tumor metastasis.

In this study, we describe an ordered formation of long- and very long-chain ceramide species in relation to the progression of B-cell receptor (BcR) triggering induced apoptosis. An early and caspase-independent increase in long-chain ceramide species, in which C-24-ceramide predominated, was observed 6 h after Belt triggering. In contrast, very long-chain ceramide species were generated later, 12-24 h after BeR triggering. The formation of these very long-chain ceramide species, in which C-24-ceramide predominated, required the activation of effector caspases. BcR-induced formation of long-chain ceramide species resulted in proteasomal activation and degradation of XIAP and subsequent activation of effector caspases, demonstrating an important cell-biological mechanism through which longchain ceramides may be involved in the progression of BcR triggering induced apoptosis and subsequent formation of very long-chain ceramide species. BcR-induced activation of the proteasome was blocked with ISP-1/myriocin, a potent and selective inhibitor of serine palmitoyl transferase that catalyzes the first and rate-limiting step in the de novo formation of ceramide. Both ISP-1 and clasto-lactacystin beta-lactone, an irreversible inhibitor of the proteasome, prevented BcR cross-linking-induced XIAP degradation. Also, a mutant XIAP lacking the ubiquitin-ligating ring finger motif was completely resistant to proteasome-mediated degradation, and Ramos cells overexpressing XIAP became highly resistant to BcR cross-linking-induced activation of caspases. The formation of C-16-ceramide in response to BcR cross-linking was found unaltered in XIAP overexpressing Ramos cells, whereas C-24-ceramide formation was completely abolished. These results demonstrate how de novo generated long-chain ceramide species may be involved in the activation of downstream effector caspases and subsequent formation of very long-chain ceramide species. As such, these results provide novel and important insights into the significance of specific ceramide species in defined stages of apoptosis.

In HepG2 cells, the subapical compartment (SAC) is involved in the biogenesis of membrane polarity. By contrast, direct apical transport originating from the trans-Golgi network (TGN), which may contribute to polarity establishment, has been poorly defined in these cells. Thus, although newly synthesized sphingolipids can be directly transported from the TGN to the apical membrane, numerous apical resident proteins are traveling via the transcytotic route. Here, we developed an in vitro transport assay and compared the molecular sorting of 6-[N-(7-nitrobenz-2-oxa-1,3 diazol-4-yl)amino] hexanoyl-sphingomyelin (C6NBD-SM) and C6NBD-glucosylceramide (C6NBD-GlcCer) in TGN and SAC. SM is released from both TGN and SAC in the lumenal leaflet of transport vesicles. This holds also for GlcCer released from the SAC but not for a substantial fraction that departed from the Golgi. Distinct transport vesicles, enriched in either SM or GlcCer are released from SAC, consistent with their rigid sorting in this compartment. Different vesicle populations could not be recovered from TGN, although in situ experiments reveal that GlcCer is preferentially transported to the apical membrane, reflecting different transport mechanisms. The results indicate that in HepG2 cells sphingolipids are mainly sorted in the SAC membrane and that the release of SM from SAC and TGN is differentially regulated.

Chronic saturated fatty acid exposure causes β-cell apoptosis and, thus, contributes to type 2 diabetes. Although endoplasmic reticulum (ER) stress and reduced ER-to-Golgi protein trafficking have been implicated, the exact mechanisms whereby saturated fatty acids trigger β-cell death remain elusive. Using mass spectroscopic lipidomics and subcellular fractionation, we demonstrate that palmitate pretreatment of MIN6 β-cells promoted ER remodeling of both phospholipids and sphingolipids, but only the latter was causally linked to lipotoxic ER stress. Thus, overexpression of glucosylceramide synthase, previously shown to protect against defective protein trafficking and ER stress, partially reversed lipotoxic reductions in ER sphingomyelin (SM) content and aggregation of ER lipid rafts, as visualized using Erlin1-GFP. Using both lipidomics and a sterol response element reporter assay, we confirmed that free cholesterol in the ER was also reciprocally modulated by chronic palmitate and glucosylceramide synthase overexpression. This is consistent with the known coregulation and association of SM and free cholesterol in lipid rafts. Inhibition of SM hydrolysis partially protected against ATF4/C/EBP homology protein induction because of palmitate. Our results suggest that loss of SM in the ER is a key event for initiating β-cell lipotoxicity, which leads to disruption of ER lipid rafts, perturbation of protein trafficking, and initiation of ER stress.

In BCR-ABL-expressing cells, sphingolipid metabolism is altered. Because the first step of sphingolipid biosynthesis occurs in the endoplasmic reticulum (ER), our objective was to identify ABL targets in the ER. A phosphoproteomic analysis of canine pancreatic ER microsomes identified 49 high scoring phosphotyrosine-containing peptides. These were then categorized in silico and validated in vitro. We demonstrated that the ER-resident human protein serine palmitoyltransferase long chain-1 (SPTLC1), which is the first enzyme of sphingolipid biosynthesis, is phosphorylated at Tyr164 by the tyrosine kinase ABL. Inhibition of BCR-ABL using either imatinib or shRNA-mediated silencing led to the activation of SPTLC1 and to increased apoptosis in both K562 and LAMA-84 cells. Finally, we demonstrated that mutation of Tyr164 to Phe in SPTLC1 increased serine palmitoyltransferase activity. The Y164F mutation also promoted the remodeling of cellular sphingolipid content, thereby sensitizing K562 cells to apoptosis. Our observations provide a mechanistic explanation for imatinib-mediated cell death and a novel avenue for therapeutic strategies. Background: Increasing evidence points toward the role of tyrosine phosphorylation in the regulation of ER homeostasis. Results: BCR-ABL-mediated tyrosine phosphorylation of SPTLC1 at Tyr164 attenuates SPT activity, thus preventing ceramide-dependent cell death. Conclusion: Tyrosine phosphorylation is critical for regulating SPT activity in the ER. Significance: This is a novel mechanism of oncogenic protein kinase-dependent control of sphingolipid metabolism.

Ceramide is a key intermediate in the pathway of sphingolipid biosynthesis and is an important intracellular messenger. We recently generated a ceramide synthase 2 (CerS2) null mouse that cannot synthesize very long acyl chain (C22-C24) ceramides. This mouse displays severe and progressive hepatopathy. Significant changes were observed in the sphingolipid profile of CerS2 null mouse liver, including elevated C16-ceramide and sphinganine levels in liver and in isolated mitochondrial fractions. Because ceramide may be involved in reactive oxygen species (ROS) formation, we examined whether ROS generation was affected in CerS2 null mice. Levels of a number of anti-oxidant enzymes were elevated, as were lipid peroxidation, protein nitrosylation, and ROS. ROS were generated from mitochondria due to impaired complex IV activity. C16-ceramide, sphingosine, and sphinganine directly inhibited complex IV activity in isolated mitochondria and in mitoplasts, whereas other ceramide species, sphingomyelin, and diacylglycerol were without effect. A fluorescent analog of sphinganine accumulated in mitochondria. Heart mitochondria did not display a substantial alteration in the sphingolipid profile or in complex IV activity. We suggest that C16-ceramide and/or sphinganine induce ROS formation through the modulation of mitochondrial complex IV activity, resulting in chronic oxidative stress. These results are of relevance for understanding modulation of ROS signaling by sphingolipids.

FoxO1 transcription factor controls the glucose and lipid metabolism, as well as cell proliferation and stress response. Akt, activated by insulin and other growth factors, phosphorylates FoxO1 causing its nuclear export and activity suppression. In this manuscript, we show that IL-1β, a pro-inflammatory cytokine, has the opposite effects on FoxO1. IL-1β stimulation of primary rat hepatocytes and HEK293 cells overexpressing the IL-1β receptor (293-IL-1RI) results in increased nuclear and cytosolic FoxO1 protein but not mRNA levels. IL-1β stimulation also elevates the levels of a mutant FoxO1 that is resistant to Akt phosphorylation. This suggests that an Akt-independent mechanism is involved. Co-stimulation with insulin does not affect the IL-1β induction of FoxO1. The IL-1β effects on FoxO1 are counteracted, however, by the silencing or inhibition of neutral sphingomyelinase 2 (nSMase-2) using shRNAi, scyphostatin, or GW4869, as well as by the pharmacological inhibition of JNK and ERK. Reversely, the overexpression of nSMase-2 through adenovirus-mediated gene transfer potentiates, in a JNK- and ERK-dependent manner, the IL-1β effects. We also show that transcription of insulin-like growth factor-binding protein-1 mRNA, which requires active FoxO1, is stimulated by IL-1β and is suppressed by the inhibition of nSMase-2 and JNK. In conclusion, we propose that IL-1β regulates FoxO1 activity through a novel nSMase-2-dependent pathway.

The brain-specific isoform carnitine palmitoyltransferase 1C (CPT1C) has been implicated in the hypothalamic regulation of food intake and energy homeostasis. Nevertheless, its molecular function is not completely understood, and its role in other brain areas is unknown. We demonstrate that CPT1C is expressed in pyramidal neurons of the hippocampus and is located in the endoplasmic reticulum throughout the neuron, even inside dendritic spines. We used molecular, cellular, and behavioral approaches to determine CPT1C function. First, we analyzed the implication of CPT1C in ceramide metabolism. CPT1C overexpression in primary hippocampal cultured neurons increased ceramide levels, whereas in CPT1C-deficient neurons, ceramide levels were diminished. Correspondingly, CPT1C knock-out (KO) mice showed reduced ceramide levels in the hippocampus. At the cellular level, CPT1C deficiency altered dendritic spine morphology by increasing immature filopodia and reducing mature mushroom and stubby spines. Total protrusion density and spine head area in mature spines were unaffected. Treatment of cultured neurons with exogenous ceramide reverted the KO phenotype, as did ectopic overexpression of CPT1C, indicating that CPT1C regulation of spine maturation is mediated by ceramide. To study the repercussions of the KO phenotype on cognition, we performed the hippocampus-dependent Morris water maze test on mice. Results show that CPT1C deficiency strongly impairs spatial learning. All of these results demonstrate that CPT1C regulates the levels of ceramide in the endoplasmic reticulum of hippocampal neurons, and this is a relevant mechanism for the correct maturation of dendritic spines and for proper spatial learning.

Toll-like receptor 4 (TLR4) is a component of the innate immune system that recognizes a diverse group of molecular structures, such as lipopolysaccharide (LPS) from Gram-negative bacteria. TLR4 signaling ultimately leads to activation of the transcription factor, nuclear factor κB (NF-κB), and the production of cytokines. Ceramide is a bioactive sphingolipid that has been suggested to regulate TLR4-induced NF-κB signaling, although reports on the role of ceramide in TLR4 activation conflict. We investigated the possibility that ceramide metabolites, such as ceramide-1-phosphate (C-1-P), may explain these discrepancies. We now report that exogenous C-1-P, but not ceramide, reduces NF-κB-mediated gene transcription in HEK 293 cells stably transfected with human TLR4, CD14, and MD-2. We demonstrate that inhibition of NF-κB by exogenous C-1-P is dose-dependent and specific to TLR4 in a reporter assay. We further demonstrate a requirement for both the phosphate moiety and the sphingoid backbone to inhibit LPS-activated NF-κB transcription. Specifically, C-1-P prevents the degradation of IκB, the phosphorylation of the p65 subunit of NF-κB, and LPS-stimulated MAPK activation. The functional consequence of C-1-P inhibition of NF-κB is a reduction in LPS-mediated cytokine release from HEK 293 TLR4-expressing cells and human peripheral blood mononuclear cells. Taken together, these data demonstrate that C-1-P may function as an anti-inflammatory lipid mediator of immune response.

Sphingosine 1-phosphate (S1P) and ceramide have been implicated in both autophagy and apoptosis. However, the roles of these sphingolipid metabolites in the links between these two processes are not completely understood. Depletion of S1P phosphohydrolase-1 (SPP1), which degrades intracellular S1P, induces the unfolded protein response and endoplasmic reticulum stress-induced autophagy (Lepine, S., Allegood, J. C., Park, M., Dent, P., Milstien, S., and Spiegel, S. (2011) Cell Death Differ. 18, 350–361). Surprisingly, however, treatment with doxorubicin, which by itself also induced autophagy, markedly reduced the extent of autophagy mediated by depletion of SPP1. Concomitantly, doxorubicin-induced apoptosis was greatly enhanced by down-regulation of SPP1. Autophagy and apoptosis seemed to be sequentially linked because inhibiting autophagy with 3-methyladenine also markedly attenuated apoptosis. Moreover, silencing Atg5 or the three sensors of the unfolded protein response, IRE1α, ATF6, and PKR-like eIF2α kinase (PERK), significantly decreased both autophagy and apoptosis. Doxorubicin stimulated calpain activity and Atg5 cleavage, which were significantly enhanced in SPP1-depleted cells. Inhibition or depletion of calpain not only suppressed Atg5 cleavage, it also markedly decreased the robust apoptosis induced by doxorubicin in SPP1-deficient cells. Importantly, doxorubicin also increased de novo synthesis of the pro-apoptotic sphingolipid metabolite ceramide. Elevation of ceramide in turn stimulated calpain; conversely, inhibiting ceramide formation suppressed Atg5 cleavage and apoptosis. Hence, doxorubicin switches protective autophagy in SPP1-depleted cells to apoptosis by calpain-mediated Atg5 cleavage.

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.

Background: Pro-TNFα is transformed into the active/soluble form through proteolysis by TNFα-converting enzyme (TACE). Results: Genetic ablation of ceramide kinase induces an increase in TACE activity and secreted TNFα. Conclusion: Ceramide 1-phosphate (C1P) negatively regulates the activity of TACE. Significance: The TACE/C1P interaction is a viable drug target for the treatment of heart disease and sepsis., Tumor necrosis factor α (TNFα) is a well known cytokine involved in systemic and acute inflammation. In this study, we demonstrate that ceramide 1-phosphate (C1P) produced by ceramide kinase (CERK) is a negative regulator of LPS-induced TNFα secretion. Specifically, bone marrow-derived macrophages isolated from CERK knock-out mice (CERK−/−) generated higher levels of TNFα than the wild-type mice (CERK+/+) in response to LPS. An increase in basal TNFα secretion was also observed in CERK−/− murine embryonic fibroblasts, which was rescued by re-expression of wild-type CERK. This effect was due to increased secretion and not transcription. The secretion of TNFα is regulated by TNFα-converting enzyme (TACE also known as ADAM17), and importantly, the activity of TACE was higher in cell extracts from CERK−/− as compared with wild type. In vitro analysis also demonstrated that C1P is a potent inhibitor of this enzyme, in stark contrast to ceramide and sphingosine 1-phosphate. Furthermore, TACE specifically bound C1P with high affinity. Finally, several putative C1P-binding sites were identified via homology throughout the protein sequence of TACE. These results indicate that C1P produced by CERK has a negative effect on the processing/secretion of TNFα via modulation of TACE activity.

Reports suggest that excessive ceramide accumulation in mitochondria is required to initiate the intrinsic apoptotic pathway and subsequent cell death, but how ceramide accumulates is unclear. Here we report that liver mitochondria exhibit ceramide formation from sphingosine and palmitoyl-CoA and from sphingosine and palmitate. Importantly, this activity was markedly decreased in liver from neutral ceramidase (NCDase)-deficient mice. Moreover, the levels of ceramide were dissimilar in liver mitochondria of WT and NCDase KO mice. These results suggest that NCDase is a key participant of ceramide formation in liver mitochondria. We also report that highly purified liver mitochondria have ceramidase, reverse ceramidase, and thioesterase activities. Increased accessibility of palmitoyl-CoA to the mitochondrial matrix with the pore-forming peptide zervamicin IIB resulted in 2-fold increases in palmitoyl-CoA hydrolysis by thioesterase. This increased hydrolysis was accompanied by an increase in ceramide formation, demonstrating that both outer membrane and matrix localized thioesterases can regulate ceramide formation. Also, ceramide formation might occur both in the outer mitochondrial membrane and in the mitochondrial matrix, suggesting the existence of distinct ceramide pools. Taken together, these results suggest that the reverse activity of NCDase contributes to sphingolipid homeostasis in this organelle in vivo.

A barrier to water loss is vital to maintaining life on dry land. Formation of the mammalian skin barrier requires both the essential fatty acid linoleate and the two lipoxygenases 12R-lipoxygenase (12R-LOX) and epidermal lipoxygenase-3 (eLOX3), although their roles are poorly understood. Linoleate occurs in O-linoleoyl-ω-hydroxyceramide, which, after hydrolysis of the linoleate moiety, is covalently attached to protein via the free ω-hydroxyl of the ceramide, forming the corneocyte lipid envelope, a scaffold between lipid and protein that helps seal the barrier. Here we show using HPLC-UV, LC-MS, GC-MS, and (1)H NMR that O-linoleoyl-ω-hydroxyceramide is oxygenated in a regio- and stereospecific fashion by the consecutive actions of 12R-LOX and eLOX3 and that these products occur naturally in pig and mouse epidermis. 12R-LOX forms 9R-hydroperoxy-linoleoyl-ω-hydroxyceramide, further converted by eLOX3 to specific epoxyalcohol (9R,10R-trans-epoxy-11E-13R-hydroxy) and 9-keto-10E,12Z esters of the ceramide; an epoxy-ketone derivative (9R,10R-trans-epoxy-11E-13-keto) is the most prominent oxidized ceramide in mouse skin. These products are absent in 12R-LOX-deficient mice, which crucially display a near total absence of protein-bound ω-hydroxyceramides and of the corneocyte lipid envelope and die shortly after birth from transepidermal water loss. We conclude that oxygenation of O-linoleoyl-ω-hydroxyceramide is required to facilitate the ester hydrolysis and allow bonding of the ω-hydroxyceramide to protein, providing a coherent explanation for the roles of multiple components in epidermal barrier function. Our study uncovers a hitherto unknown biochemical pathway in which the enzymic oxygenation of ceramides is involved in building a crucial structure of the epidermal barrier.

The sphingolipid ceramide has been widely implicated in the regulation of programmed cell death or apoptosis. The accumulation of ceramide has been demonstrated in a wide variety of experimental models of apoptosis and in response to a myriad of stimuli and cellular stresses. However, the detailed mechanisms of its generation and regulatory role during apoptosis are poorly understood. We sought to determine the regulation and roles of ceramide production in a model of ultraviolet light-C (UV-C)-induced programmed cell death. We found that UV-C irradiation induces the accumulation of multiple sphingolipid species including ceramide, dihydroceramide, sphingomyelin, and hexosylceramide. Late ceramide generation was also found to be regulated by Bcl-xL, Bak, and caspases. Surprisingly, inhibition of de novo synthesis using myriocin or fumonisin B1 resulted in decreased overall cellular ceramide levels basally and in response to UV-C, but only fumonisin B1 inhibited cell death, suggesting the presence of a ceramide synthase (CerS)-dependent, sphingosine-derived pool of ceramide in regulating programmed cell death. We found that this pool did not regulate the mitochondrial pathway, but it did partially regulate activation of caspase-7 and, more importantly, was necessary for late plasma membrane permeabilization. Attempting to identify the CerS responsible for this effect, we found that combined knockdown of CerS5 and CerS6 was able to decrease long-chain ceramide accumulation and plasma membrane permeabilization. These data identify a novel role for CerS and the sphingosine salvage pathway in regulating membrane permeability in the execution phase of programmed cell death.

Glycolipid transfer protein (GLTP) accelerates glycolipid intermembrane transfer via a unique lipid transfer/binding fold (GLTP fold) that defines the GLTP superfamily and is the prototype for functional GLTP-like domains in larger proteins, i.e. FAPP2. Human GLTP is encoded by the single-copy GLTP gene on chromosome 12 (12q24.11 locus), but regulation of GLTP gene expression remains completely unexplored. Herein, the ability of glycosphingolipids (and their sphingolipid metabolites) to regulate the transcriptional expression of GLTP via its promoter has been evaluated. Using luciferase and GFP reporters in concert with deletion mutants, the constitutive and basal (225 bp; ∼78% G+C) human GLTP promoters have been defined along with adjacent regulatory elements. Despite high G+C content, translational regulation was not evident by the mammalian target of rapamycin pathway. Four GC-boxes were shown to be functional Sp1/Sp3 transcription factor binding sites. Mutation of one GC-box was particularly detrimental to GLTP transcriptional activity. Sp1/Sp3 RNA silencing and mithramycin A treatment significantly inhibited GLTP promoter activity. Among tested sphingolipid analogs of glucosylceramide, sulfatide, ganglioside GM1, ceramide 1-phosphate, sphingosine 1-phosphate, dihydroceramide, sphingosine, only ceramide, a nonglycosylated precursor metabolite unable to bind to GLTP protein, induced GLTP promoter activity and raised transcript levels in vivo. Ceramide treatment partially blocked promoter activity decreases induced by Sp1/Sp3 knockdown. Ceramide treatment also altered the in vivo binding affinity of Sp1 and Sp3 for the GLTP promoter and decreased Sp3 acetylation. This study represents the first characterization of any Gltp gene promoter and links human GLTP expression to sphingolipid homeostasis through ceramide.

A decreased clearance of apoptotic cells (efferocytosis) by alveolar macrophages (AM) may contribute to inflammation in emphysema. The up-regulation of ceramides in response to cigarette smoking (CS) has been linked to AM accumulation and increased detection of apoptotic alveolar epithelial and endothelial cells in lung parenchyma. We hypothesized that ceramides inhibit the AM phagocytosis of apoptotic cells. Release of endogenous ceramides via sphingomyelinase or exogenous ceramide treatments dose-dependently impaired apoptotic Jurkat cell phagocytosis by primary rat or human AM, irrespective of the molecular species of ceramide. Similarly, in vivo augmentation of lung ceramides via intratracheal instillation in rats significantly decreased the engulfment of instilled target apoptotic thymocytes by resident AM. The mechanism of ceramide-induced efferocytosis impairment was dependent on generation of sphingosine via ceramidase. Sphingosine treatment recapitulated the effects of ceramide, dose-dependently inhibiting apoptotic cell clearance. The effect of ceramide on efferocytosis was associated with decreased membrane ruffle formation and attenuated Rac1 plasma membrane recruitment. Constitutively active Rac1 overexpression rescued AM efferocytosis against the effects of ceramide. CS exposure significantly increased AM ceramides and recapitulated the effect of ceramides on Rac1 membrane recruitment in a sphingosine-dependent manner. Importantly, CS profoundly inhibited AM efferocytosis via ceramide-dependent sphingosine production. These results suggest that excessive lung ceramides may amplify lung injury in emphysema by causing both apoptosis of structural cells and inhibition of their clearance by AM.

The acid sphingomyelinase (aSMase) gene gives rise to two distinct enzymes, lysosomal sphingomyelinase (L-SMase) and secretory sphingomyelinase (S-SMase), via differential trafficking of a common protein precursor. However, the regulation of S-SMase and its role in cytokine-induced ceramide formation remain ill defined. To determine the role of S-SMase in cellular sphingolipid metabolism, MCF7 breast carcinoma cells stably transfected with V5-aSMase(WT) were treated with inflammatory cytokines. Interleukin-1β and tumor necrosis factor-α induced a time- and dose-dependent increase in S-SMase secretion and activity, coincident with selective elevations in cellular C(16)-ceramide. To establish a role for S-SMase, we utilized a mutant of aSMase (S508A) that is shown to retain L-SMase activity, but is defective in secretion. MCF7 expressing V5-aSMase(WT) exhibited increased S-SMase and L-SMase activity, as well as elevated cellular levels of specific long-chain and very long-chain ceramide species relative to vector control MCF7. Interestingly, elevated levels of only certain very long-chain ceramides were evident in V5-aSMase(S508A) MCF7. Secretion of the S508A mutant was also defective in response to IL-1β, as was the regulated generation of C(16)-ceramide. Taken together, these data support a crucial role for Ser(508) in the regulation of S-SMase secretion, and they suggest distinct metabolic roles for S-SMase and L-SMase.

ERM proteins are regulated by phosphorylation of the most C-terminal threonine residue, switching them from an activated to an inactivated form. However, little is known about the control of this regulation. Previous work in our group demonstrated that secretion of acid sphingomyelinase acts upstream of ERM dephosphorylation, suggesting the involvement of sphingomyelin (SM) hydrolysis in ERM regulation. To define the role of specific lipids, we employed recombinant bacterial sphingomyelinase (bSMase) as a direct probe of SM metabolism at the plasma membrane. bSMase induced a rapid dose- and time-dependent decrease in ERM dephosphorylation. ERM dephosphorylation was driven by ceramide generation and not by sphingomyelin depletion, as shown using recombinant sphingomyelinase D. The generation of ceramide at the plasma membrane was sufficient for ERM regulation, and no intracellular SM hydrolysis was required, as was visualized using Venus-tagged lysenin probe, which specifically binds SM. Interestingly, hydrolysis of plasma membrane bSMase-induced ceramide using bacterial ceramidase caused ERM hyperphosphorylation and formation of cell surface protrusions. The effects of plasma membrane ceramide hydrolysis were due to sphingosine 1-phosphate formation, as ERM phosphorylation was blocked by an inhibitor of sphingosine kinase and induced by sphingosine 1-phosphate. Taken together, these results demonstrate a new regulatory mechanism of ERM phosphorylation by sphingolipids with opposing actions of ceramide and sphingosine 1-phosphate. The approach also defines a tool kit to probe sphingolipid signaling at the plasma membrane.

Increased generation of dihydrosphingosine (DHS), a bioactive sphingolipid, has been implicated in the cytotoxicity of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) in tumor cells. However, how 4-HPR increases DHS remains unclear. Here we demonstrate that 4-HPR increases the expression of ACER2, which catalyzes the hydrolysis of dihydroceramides to generate DHS, and that ACER2 up-regulation plays a key role in mediating the 4-HPR-induced generation of DHS as well as the cytotoxicity of 4-HPR in tumor cells. Treatment with 4-HPR induced the accumulation of dihydroceramides (DHCs) in tumor cells by inhibiting dihydroceramide desaturase (DES) activity, which catalyzes the conversion of DHCs to ceramides. Treatment with 4-HPR also increased ACER2 expression through a retinoic acid receptor-independent and caspase-dependent manner. Overexpression of ACER2 augmented the 4-HPR-induced generation of DHS as well as 4-HPR cytotoxicity, and 4-HPR-induced death in tumor cells, whereas knocking down ACER2 had the opposite effects. ACER2 overexpression, along with treatment with GT11, another DES inhibitor, markedly increased cellular DHS, leading to tumor cell death, whereas ACER2 overexpression or GT11 treatment alone failed to do so, suggesting that both ACER2 up-regulation and DES inhibition are necessary and sufficient to mediate 4-HPR-induced DHS accumulation, cytotoxicity, and death in tumor cells. Taken together, these results suggest that up-regulation of the ACER2/DHS pathway mediates the cytotoxicity of 4-HPR in tumor cells and that up-regulating or activating ACER2 may improve the anti-cancer activity of 4-HRR and other DHC-inducing agents.

The pathogenesis and treatment of nonalcoholic steatohepatitis (NASH) are not well established. Feeding a diet deficient in both methionine and choline (MCD) is one of the most common models of NASH, which is characterized by steatosis, mitochondrial dysfunction, hepatocellular injury, oxidative stress, inflammation, and fibrosis. However, the individual contribution of the lack of methionine and choline in liver steatosis, advanced pathology and impact on mitochondrial S-adenosyl-l-methionine (SAM) and glutathione (GSH), known regulators of disease progression, has not been specifically addressed. Here, we examined the regulation of mitochondrial SAM and GSH and signs of disease in mice fed a MCD, methionine-deficient (MD), or choline-deficient (CD) diet. The MD diet reproduced most of the deleterious effects of MCD feeding, including weight loss, hepatocellular injury, oxidative stress, inflammation, and fibrosis, whereas CD feeding was mainly responsible for steatosis, characterized by triglycerides and free fatty acids accumulation. These findings were preceded by MCD- or MD-mediated SAM and GSH depletion in mitochondria due to decreased mitochondrial membrane fluidity associated with a lower phosphatidylcholine/phosphatidylethanolamine ratio. MCD and MD but not CD feeding resulted in increased ceramide levels by acid sphingomyelinase. Moreover, GSH ethyl ester or SAM therapy restored mitochondrial GSH and ameliorated hepatocellular injury in mice fed a MCD or MD diet. Thus, the depletion of SAM and GSH in mitochondria is an early event in the MCD model of NASH, which is determined by the lack of methionine. Moreover, therapy using permeable GSH prodrugs may be of relevance in NASH.

Acid sphingomyelinase (ASMase) has been proposed to mediate lipopolysaccharide (LPS) signaling in various cell types. This study shows that ASMase is a negative regulator of LPS-induced tumor necrosis factor alpha (TNFalpha) secretion in macrophages. ASMase-deficient (asm(-/-)) mice and isolated peritoneal macrophages produce severalfold more TNFalpha than their wild-type (asm(+/+)) counterparts when stimulated with LPS, whereas the addition of exogenous ceramides or sphingomyelinase reduces the differences. The underlying mechanism for these effects is not transcriptional but post-translational. The TNFalpha-converting enzyme (TACE) catalyzes the maturation of the 26-kDa precursor (pro-TNFalpha) to an active 17-kDa form (soluble (s)TNFalpha). In mouse peritoneal macrophages, the activity of TACE was the rate-limiting factor regulating TNFalpha production. A substantial portion of the translated pro-TNFalpha was not processed to sTNFalpha; instead, it was rapidly internalized and degraded in the lysosomes. TACE activity was 2-3-fold higher in asm(-/-) macrophages as compared with asm(+/+) macrophages and was suppressed when cells were treated with exogenous ceramide and sphingomyelinase. Indirect immunofluorescence analyses revealed distinct TNFalpha-positive structures in the close vicinity of the plasma membrane in asm(-/-) but not in asm(+/+) macrophages. asm(-/-) cells also had a higher number of early endosomal antigen 1-positive early endosomes. Experiments that involved inhibitors of TACE, endocytosis, and lysosomal proteolysis suggest that in the asm(-/-) cells a significant portion of pro-TNFalpha was sequestered within the early endosomes, and instead of undergoing lysosomal proteolysis, it was recycled to the plasma membrane and processed to sTNFalpha.

Both cultured neonatal rat hippocampal neurons and differentiated oligodendrocytes rapidly metabolized exogenous C(2)- and C(6)-ceramides to sphingosine (Sph) and sphingosine 1-phosphate (S1P) but only minimally to C(16-24)-ceramides. Dihydrosphinolipids were unaffected but were increased by exogenous C(6)-dihydroceramide. Conversely, quantitative liquid chromatography-tandem mass spectrometry technology showed that exogenous S1P (0.25-10 microm) was rapidly metabolized to both Sph (a200-fold increase) and predominantly C(18)-ceramide (a2-fold increase). Longer treatments with either C(2)-ceramide (2.5 microm) or S1P (10 microm) led to apoptotic cell death. Thus, there is an active sphingolipid salvage pathway in both neurons and oligodendrocytes. Staurosporine-induced cell death was shown to be associated with decreased S1P and increased Sph and C(16/18)-ceramide levels. The physiological significance of this observation was confirmed by the analysis of affected white matter and plaques from brains of multiple sclerosis patients in which reduced S1P and increased Sph and C(16/18)-ceramides were observed.

Ceramide is an important lipid signaling molecule that plays critical roles in regulating cell behavior. Ceramide synthesis is surprisingly complex and is orchestrated by six mammalian ceramide synthases, each of which produces ceramides with restricted acyl chain lengths. We have generated a CerS2 null mouse and characterized the changes in the long chain base and sphingolipid composition of livers from these mice. Ceramide and downstream sphingolipids were devoid of very long (C22–C24) acyl chains, consistent with the substrate specificity of CerS2 toward acyl-CoAs. Unexpectedly, C16-ceramide levels were elevated, and as a result, total ceramide levels were unaltered; however, C16-ceramide synthesis in vitro was not increased. Levels of sphinganine were also significantly elevated, by up to 50-fold, reminiscent of the effect of the ceramide synthase inhibitor, fumonisin B1. With the exceptions of glucosylceramide synthase and neutral sphingomyelinase 2, none of the other enzymes tested in either the sphingolipid biosynthetic or degradative pathways were significantly changed. Total glycerophospholipid and cholesterol levels were unaltered, although there was a marked elevation in C18:1 and C18:2 fatty acids in phosphatidylethanolamine, concomitant with a reduction in C18:0 and C20:4 fatty acids. Finally, differences were observed in the biophysical properties of lipid extracts isolated from liver microsomes, with membranes from CerS2 null mice displaying higher membrane fluidity and showing morphological changes. Together, these results demonstrate novel modes of cross-talk and regulation between the various branches of lipid metabolic pathways upon inhibition of very long acyl chain ceramide synthesis.

Ceramides with different fatty acyl chains may vary in their physiological or pathological roles; however, it remains unclear how cellular levels of individual ceramide species are regulated. Here, we demonstrate that our previously cloned human alkaline ceramidase 3 (ACER3) specifically controls the hydrolysis of ceramides carrying unsaturated long acyl chains, unsaturated long-chain (ULC) ceramides. In vitro, ACER3 only hydrolyzed C(18:1)-, C(20:1)-, C(20:4)-ceramides, dihydroceramides, and phytoceramides. In cells, ACER3 overexpression decreased C(18:1)- and C(20:1)-ceramides and dihydroceramides, whereas ACER3 knockdown by RNA interference had the opposite effect, suggesting that ACER3 controls the catabolism of ULC ceramides and dihydroceramides. ACER3 knockdown inhibited cell proliferation and up-regulated the cyclin-dependent kinase inhibitor p21(CIP1/WAF1). Blocking p21(CIP1/WAF1) up-regulation attenuated the inhibitory effect of ACER3 knockdown on cell proliferation, suggesting that ACER3 knockdown inhibits cell proliferation because of p21(CIP1/WAF1) up-regulation. ACER3 knockdown inhibited cell apoptosis in response to serum deprivation. ACER3 knockdown up-regulated the expression of the alkaline ceramidase 2 (ACER2), and the ACER2 up-regulation decreased non-ULC ceramide species while increasing both sphingosine and its phosphate. Collectively, these data suggest that ACER3 catalyzes the hydrolysis of ULC ceramides and dihydroceramides and that ACER3 coordinates with ACER2 to regulate cell proliferation and survival.

Intracellular lipid accumulation in the heart is associated with cardiomyopathy, yet the precise role of triglyceride (TG) remains unclear. With exercise, wild type hearts develop physiologic hypertrophy. This was associated with greater TG stores and a marked induction of the TG-synthesizing enzyme diacylglycerol (DAG) acyltransferase 1 (DGAT1). Transgenic overexpression of DGAT1 in the heart using the cardiomyocyte- specific alpha-myosin heavy chain (MHC) promoter led to approximately a doubling of DGAT activity and TG content and reductions of approximately 35% in cardiac ceramide, 26% in DAG, and 20% in free fatty acid levels. Cardiac function assessed by echocardiography and cardiac catheterization was unaffected. These mice were then crossed with animals expressing long-chain acyl-CoA synthetase via the MHC promoter (MHC-ACS), which develop lipotoxic cardiomyopathy. MHC-DGAT1XMHC-ACS double transgenic male mice had improved heart function; fractional shortening increased by 74%, and diastolic function improved compared with MHC-ACS mice. The improvement of heart function correlated with a reduction in cardiac DAG and ceramide and reduced cardiomyocyte apoptosis but increased fatty acid oxidation. In addition, the survival of the mice was improved. Our study indicates that TG is not likely to be a toxic lipid species directly, but rather it is a feature of physiologic hypertrophy and may serve a cytoprotective role in lipid overload states. Moreover, induction of DGAT1 could be beneficial in the setting of excess heart accumulation of toxic lipids.

FTY720, a sphingosine analog, is in clinical trials as an immunomodulator. The biological effects of FTY720 are believed to occur after its metabolism to FTY720 phosphate. However, very little is known about whether FTY720 can interact with and modulate the activity of other enzymes of sphingolipid metabolism. We examined the ability of FTY720 to modulate de novo ceramide synthesis. In mammals, ceramide is synthesized by a family of six ceramide synthases, each of which utilizes a restricted subset of acyl-CoAs. We show that FTY720 inhibits ceramide synthase activity in vitro by noncompetitive inhibition toward acyl-CoA and uncompetitive inhibition toward sphinganine; surprisingly, the efficacy of inhibition depends on the acyl-CoA chain length. In cultured cells, FTY720 has a more complex effect, with ceramide synthesis inhibited at high (500 nm to 5 μm) but not low (

Atypical protein kinase Cs (PKCs) (aPKCzeta and lambda/iota) have emerged as important binding partners for ceramide, a membrane-resident cell signaling lipid that is involved in the regulation of apoptosis as well as cell polarity. Using ceramide overlay assays with proteolytic fragments of PKCzeta and vesicle binding assays with ectopically expressed protein, we show that a protein fragment comprising the carboxyl-terminal 20-kDa sequence of PKCzeta (C20zeta, amino acids 405-592) bound to C16:0 ceramide. This sequence is not identical to the C1 domain (amino acids 131-180), which has been suggested to serve as a potential ceramide binding domain. Using immunocytochemistry, we found that a C20zeta protein fragment ectopically expressed in two epithelial cell types (neural progenitors and Madin-Darby canine kidney cells) co-distributed with ceramide. Stable expression of C20zeta-EGFP in Madin-Darby canine kidney cells disrupted the formation of adherens and tight junctions and impaired the epithelium integrity by reducing transepithelial electrical resistance. Disruption of cell adhesion and loss of transepithelial electrical resistance was prevented by incubation with C16:0 ceramide. Our results show, for the first time, that there is a novel ceramide binding domain (C20zeta) in the carboxyl terminus of aPKC. Our results also show that the interaction of ceramide with this binding domain is essential for cell-to-cell contacts in epithelia. Therefore, ceramide interaction with the C20zeta binding domain is a potential mechanism by which ceramide and aPKC regulate the formation of junctional complexes in epithelial cells.

Novel immunomodulatory molecule FTY720 is a synthetic analog of myriocin, but unlike myriocin FTY720 does not inhibit serine palmitoyltransferase. Although many of the effects of FTY720 are ascribed to its phosphorylation and subsequent sphingosine 1-phosphate (S1P)-like action through S1P1,3–5 receptors, studies on modulation of intracellular balance of signaling sphingolipids by FTY720 are limited. In this study, we used stable isotope pulse labeling of human pulmonary artery endothelial cells with l-[U-13C, 15N]serine as well as in vitro enzymatic assays and liquid chromatography-tandem mass spectrometry methodology to characterize FTY720 interference with sphingolipid de novo biosynthesis. In human pulmonary artery endothelial cells, FTY720 inhibited ceramide synthases, resulting in decreased cellular levels of dihydroceramides, ceramides, sphingosine, and S1P but increased levels of dihydrosphingosine and dihydrosphingosine 1-phosphate (DHS1P). The FTY720-induced modulation of sphingolipid de novo biosynthesis was similar to that of fumonisin B1, a classical inhibitor of ceramide synthases, but differed in the efficiency to inhibit biosynthesis of short-chain versus long-chain ceramides. In vitro kinetic studies revealed that FTY720 is a competitive inhibitor of ceramide synthase 2 toward dihydrosphingosine with an apparent Ki of 2.15 μm. FTY720-induced up-regulation of DHS1P level was mediated by sphingosine kinase (SphK) 1, but not SphK2, as confirmed by experiments using SphK1/2 silencing with small interfering RNA. Our data demonstrate for the first time the ability of FTY720 to inhibit ceramide synthases and modulate the intracellular balance of signaling sphingolipids. These findings open a novel direction for therapeutic applications of FTY720 that focuses on inhibition of ceramide biosynthesis, ceramide-dependent signaling, and the up-regulation of DHS1P generation in cells.

Different lines of evidence indicate that both aberrant activation of the phosphatidylinositol 3-OH kinase (PI3K)/Akt survival pathway and down-regulation of the death mediator ceramide play a critical role in the aggressive behavior, apoptosis resistance, and adverse clinical outcome of glioblastoma multiforme. Furthermore, the inhibition of the PI3K/Akt pathway and the up-regulation of ceramide have been found functional to the activity of many cytotoxic treatments against glioma cell lines and glioblastomas as well. A reciprocal control between PI3K/Akt and ceramide signaling in glioma cell survival/death is suggested by data demonstrating a protective role of PI3K/Akt on ceramide-induced cell death in glial cells. In this study we investigated the role of the PI3K/Akt pathway in the regulation of the ceramide metabolism in C6 glioma cells, a cell line in which the PI3K/Akt pathway is constitutively activated. Metabolic experiments performed with different radioactive metabolic precursors of sphingolipids and microscopy studies with fluorescent ceramides demonstrated that the chemical inhibition of PI3K and the transfection with a dominant negative Akt strongly inhibited ceramide utilization for the biosynthesis of complex sphingolipids by controlling the endoplasmic reticulum (ER) to Golgi vesicular transport of ceramide. These findings constitute the first evidence for a PI3K/Akt-dependent regulation of vesicle-mediated movements of ceramide in the ER-Golgi district. Moreover, the findings also suggest the activation of the PI3K/Akt pathway as crucial to coordinate the biosynthesis of membrane complex sphingolipids with cell proliferation and growth and/or to maintain low ceramide levels, especially as concerns those treatments that promote ceramide biosynthesis in the ER.

We have recently identified a novel form of post-translational regulation of BACE1 (β-site amyloid precursor protein-cleaving enzyme 1), a membrane protein that acts as the rate-limiting enzyme in the generation of the Alzheimer disease amyloid β-peptide. Specifically, nascent BACE1 is transiently acetylated in seven lysine residues clustered in a highly disordered region of the protein that faces the lumen of the endoplasmic reticulum (ER)/ER Golgi intermediate compartment (ER/ERGIC). The acetylation protects the nascent protein from degradation by PCSK9/NARC-1 in the ERGIC and allows it to reach the Golgi apparatus. Here we report the identification of two ER/ERGIC-based acetyltransferases, ATase1 and ATase2. Both proteins display acetyl-CoA:lysine acetyltransferase activity, can interact with and acetylate BACE1, and display an ER/ERGIC localization with the catalytic site facing the lumen of the organelle. Both ATase1 and ATase2 regulate the steady-state levels of BACE1 and the rate of amyloid β-peptide generation. Finally, their transcripts are up-regulated by ceramide treatment. In conclusion, our studies have identified two new enzymes that may be involved in the pathogenesis of late-onset Alzheimer disease. The biochemical characterization of the above events could lead to the identification of novel pharmacological strategies for the prevention of this form of dementia.

Endoplasmic reticulum (ER) stress induces INS-1 cell apoptosis by a pathway involving Ca(2+)-independent phospholipase A(2) (iPLA(2)beta)-mediated ceramide generation, but the mechanism by which iPLA(2)beta and ceramides contribute to apoptosis is not well understood. We report here that both caspase-12 and caspase-3 are activated in INS-1 cells following induction of ER stress with thapsigargin, but only caspase-3 cleavage is amplified in iPLA(2)beta overexpressing INS-1 cells (OE), relative to empty vector-transfected cells, and is suppressed by iPLA(2)beta inhibition. ER stress also led to the release of cytochrome c and Smac and, unexpectedly, their accumulation in the cytosol is amplified in OE cells. These findings raise the likelihood that iPLA(2)beta participates in ER stress-induced apoptosis by activating the intrinsic apoptotic pathway. Consistent with this possibility, we find that ER stress promotes iPLA(2)beta accumulation in the mitochondria, opening of mitochondrial permeability transition pore, and loss in mitochondrial membrane potential (Delta Psi) in INS-1 cells and that these changes are amplified in OE cells. ER stress also led to greater ceramide generation in ER and mitochondria fractions of OE cells. Exposure to ceramide alone induces loss in Delta Psi and apoptosis and these are suppressed by forskolin. ER stress-induced mitochondrial dysfunction and apoptosis are also inhibited by forskolin, as well as by inactivation of iPLA(2)beta or NSMase, suggesting that iPLA(2)beta-mediated generation of ceramides via sphingomyelin hydrolysis during ER stress affect the mitochondria. In support, inhibition of iPLA(2)beta or NSMase prevents cytochrome c release. Collectively, our findings indicate that the iPLA(2)beta-ceramide axis plays a critical role in activating the mitochondrial apoptotic pathway in insulin-secreting cells during ER stress.

The present report was addressed to study the influence of sphingolipid metabolism in determining cellular fate. In nonstimulated proliferating Madin-Darby canine kidney (MDCK) cells, sphingolipid de novo synthesis is branched mainly to a production of sphingomyelin and ceramide, with a minor production of sphingosylphosphocholine, ceramide 1-phosphate, and sphingosine 1-phosphate. Experiments with 32P as a radioactive precursor showed that sphingosine 1-phosphate is produced mainly by a de novo independent pathway. Enzymatic inhibition of the de novo pathway and ceramide synthesis affected cell number and viability only slightly, without changing sphingosine 1-phosphate production. By contrast, inhibition of sphingosine kinase-1 activity provoked a significant reduction in both cell number and viability in a dose-dependent manner. When sphingolipid metabolism was studied, an increase in de novo formed ceramide was found, which correlated with the concentration of enzyme inhibitor and the reduction in cell number and viability. Knockdown of sphingosine kinase-1 expression also induced an accumulation of de novo synthesized ceramide, provoking a slight reduction in cell number and viability similar to that induced by a low concentration of the sphingosine kinase inhibitor. Taken together, our results indicate that the level of de novo formed ceramide is controlled by the synthesis of sphingosine 1-phosphate, which appears to occur through a de novo synthesis-independent pathway, most probably the salvage pathway, that is responsible for the MDCK cell fate, suggesting that under proliferating conditions, a dynamic interplay exists between the de novo synthesis and the salvage pathway.

The sphingolipid ceramide has been implicated in mediating cell death that is accompanied by mitochondrial functional alterations. Moreover, ceramide has been shown to accumulate in mitochondria upon induction of apoptotic processes. In this study, we sought to evaluate the effects of natural, highly hydrophobic long-chain ceramides on mitochondrial function in vitro. Ceramide in a dodecane/ethanol delivery system inhibited the opening of the mitochondrial permeability transition pore (PTP) induced by either oxidative stress, SH group cross-linking, or high Ca2+ load, suggesting that the inhibitory point is at a level at which major PTP regulatory pathways converge. Moreover, ceramide had no effect on well known mitochondrial components that modulate PTP activity, such as cyclophilin D, voltage-dependent anion channel, adenine nucleotide transporter, and ATP synthase. The inhibitory effect of ceramide on PTP was not stereospecific, nor was there a preference for ceramide over dihydroceramide. However, the effect of ceramide on PTP was significantly influenced by the fatty acid moiety chain length. These studies are the first to show that long-chain ceramide can influence PTP at physiologically relevant concentrations, suggesting that it is the only known potent natural inhibitor of PTP. These results suggest a novel mechanism of ceramide regulation of mitochondrial function.

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 1-phosphate (C1P) has been characterized as a sphingolipid that participates in cell signaling. Although C1P synthesis is thought to occur via phosphorylation of ceramide by ceramide kinase (CerK), the processes that regulate C1P formation and fate remain largely unknown. In this study we analyzed bone marrow-derived macrophages (BMDM) from CerK-null mice (Cerk(-/-)) and found significant levels of C1P, suggesting that previously unrecognized pathways may also lead to C1P formation. After these experiments we used an overexpression system, BMDM from Cerk(-/-) mice, and short-chain fluorescent ceramides to trace CerK-dependent formation of C1P. Because the ceramide analogs can also be converted to glucosylceramide (GlcCer) and sphingomyelin (SM), they allowed us to directly compare all three metabolites. We found that C1P produced by CerK is turned over rapidly when serum is removed or upon calcium chelation, whereas GlcCer and SM are stable under these conditions. We further demonstrated that ceramide must be transported to the Golgi complex to be phosphorylated by CerK. Inhibition of the ceramide transfer protein slowed down SM formation without decreasing C1P, suggesting an alternate route of ceramide transport. Other experiments indicated that, like GlcCer and SM, C1P traffics along the secretory pathway to reach the plasma membrane. Furthermore, in BMDM C1P was secreted more readily than was GlcCer or SM. Altogether, our results indicate that CerK is essential to C1P formation via phosphorylation of Cer, providing the first insights into mechanisms underlying ceramide access to CerK and C1P trafficking as well as clarifying C1P as a signaling entity.

Although in rat hepatocytes CD95 is predominantly located inside the cell with almost undetectable immunostaining at the plasma membrane, the addition of CD95-ligand (CD95L) induces hepatocyte apoptosis, which is preceded by a targeting and activation of intracellularly localized CD95 to the plasma membrane including formation of the death-inducing signaling complex. This process involves an NADPH oxidase-dependent generation of reactive oxygen species (ROS) through a ceramide- and protein kinase Czeta-dependent pathway, which leads to an activating phosphorylation of p47(phox). The mechanisms underlying CD95L-induced ceramide formation were addressed in the present study. It was found that CD95L lowered within seconds the apparent vesicular pH from 6.0 to 5.7 in a fluorescein isothiocyanate-dextran-accessible endosomal compartment, which was previously shown to contain acidic sphingomyelinase, and decreased N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide fluorescence, suggestive for an increase of cytosolic [Cl(-)]. Bafilomycin or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid disodium salt largely abolished the CD95L-induced endosomal acidification, ceramide formation, and downstream events, such as p47(phox) phosphorylation, ROS formation, CD95 activation, and apoptosis. These responses were also abolished after knock-down of acidic sphingomyelinase in rat hepatocytes. Interestingly, caspase 8 inhibitors abolished these CD95L-induced signaling events, including the increase in cytosolic [Cl(-)], endosomal acidification, ceramide formation, and ROS generation as well as CD95 targeting to the plasma membrane and CD95 activation. The data suggest that CD95L initiates a rapid caspase 8-dependent endosomal acidification, which triggers ceramide-dependent ROS formation as an upstream event of trafficking of intracellularly stored CD95 to the plasma membrane. It is concluded that a rapid caspase 8 activation in response to CD95L signals to intracellularly stored CD95, which becomes activated and targeted to the plasma membrane. This autoamplification of CD95-activation is required for apoptosis induction.

Programmed cell death (PCD) is involved in a variety of biologic events. Based on the morphologic appearance of the cells, there are two types of PCD as follows: apoptotic (type I) and autophagic (type II). However, the molecular machinery that determines the type of PCD is poorly defined. The purpose of this study was to show whether the presence of the cyclin-dependent kinase (CDK) inhibitor p21(WAF1/CIP1), a modulator of apoptosis, determines which type of PCD the cell undergoes. Treatment with C(2)-ceramide was associated with both the cleavage of caspase-3 and poly(ADP-ribose) polymerase and the degradation of autophagy-related Beclin 1 and Atg5 proteins, without a change in the cyclin-CDK activity, which culminated in apoptosis in p21(+/+) mouse embryonic fibroblasts (MEFs). On the other hand, C(2)-ceramide did not cleave caspase-3 or poly(ADP-ribose) polymerase and kept Beclin 1 and Atg5 proteins stable in p21(-/-) MEFs, events that this time culminated in autophagy. When expression of the p21 protein was inhibited by small interfering RNA or when the overexpression of Beclin 1 or Atg5 was induced, autophagy rather than apoptosis was initiated in the p21(+/+) MEFs treated with C(2)-ceramide. In contrast, the exogenous expression of p21 or the silencing of Beclin 1 and Atg5 with small interfering RNA increased the number of apoptotic cells and decreased the number of autophagic cells among C(2)-ceramide-treated p21(-/-) MEFs. gamma-Irradiation, which endogenously generates ceramide, induced a similar tendency in these MEFs. These results suggest that p21 plays an essential role in determining the type of cell death, positively for apoptosis and negatively for autophagy.

Hepatocyte lipoapoptosis, a critical feature of nonalcoholic steatohepatitis, can be replicated in vitro by incubating hepatocytes with saturated free fatty acids (FFA). These toxic FFA induce Bim expression, which is requisite for their cytotoxicity. Because the FoxO3a transcription factor has been implicated in Bim expression, our aim was to determine if FFA induce Bim by a FoxO3a-dependent mechanism. In Huh-7 cells, the saturated FFA, palmitic and stearic acid, increased Bim mRNA 16-fold. Treatment of cells with the saturated FFA induced FoxO3a dephosphorylation (activation) and nuclear translocation and stimulated a FoxO luciferase-based reporter assay; direct binding of FoxO3a to the Bim promoter was also confirmed by a chromatin immunoprecipitation assay. A small interfering RNA-targeted knockdown of FoxO3a abrogated FFA-mediated Bim induction and apoptosis. FoxO3a was activated by a phosphatase 2A-dependent mechanism, since okadaic acid- and small interfering RNA-targeted knockdown of this phosphatase blocked FoxO3a dephosphorylation, Bim expression, and apoptosis. Consistent with these data, phosphatase 2A activity was also stimulated 3-fold by saturated FFA. Immunoprecpitation studies revealed an FFA-dependent association between FoxO3a and protein phosphatase 2A. FFA-mediated FoxO3a activation by protein phosphatase 2A was also observed in HepG2 cells and murine hepatocytes. In conclusion, saturated FFA stimulate protein phosphatase 2A activity, which activates FoxO3a, inducing expression of the intracellular death mediator Bim.