Ceramide metabolism analysis in a model of binge drinking reveals both neuroprotective and toxic effects of ethanol

Binge drinking is a common form of alcohol abuse that involves repeated rounds of intoxication followed by withdrawal. The episodic effects of binge drinking and withdrawal on brain resident cells are thought to contribute to neural remodeling and neurological damage. However, the molecular mechanisms for these neurodegenerative effects are not understood. Ethanol (EtOH) regulates the metabolism of ceramide, a highly bioactive lipid that is enriched in brain. We used a mouse model of binge drinking to determine the effects of EtOH intoxication and withdrawal on brain ceramide metabolism. Intoxication and acute alcohol withdrawal were each associated with distinct changes in ceramide regulatory genes and metabolic products. EtOH intoxication was accompanied by decreased concentrations of multiple ceramides, coincident with reductions in the expression of enzymes involved in the production of ceramides, and increased expression of ceramide‐degrading enzymes. EtOH withdrawal was associated with specific increases in ceramide C16:0, C18:0, and C20:0 and increased expression of enzymes involved with ceramide production. These data suggest that EtOH intoxication may evoke a ceramide phenotype that is neuroprotective, whereas EtOH withdrawal results in a metabolic shift that increases the production of potentially toxic ceramide species.

     

Cardiomyocyte specific deficiency of serine palmitoyltransferase subunit 2 reduces ceramide but leads to cardiac dysfunction

The role of serine palmitoyltransferase (SPT) and de novo ceramide biosynthesis in cardiac ceramide and sphingomyelin metabolism is unclear. To determine whether the de novo synthetic pathways, rather than ceramide uptake from circulating lipoproteins, is important for heart ceramide levels, we created cardiomyocyte-specific deficiency of Sptlc2, a subunit of SPT. Heart-specific Sptlc2-deficient (hSptlc2 KO) mice had a >35% reduction in ceramide, which was limited to C18:0 and very long chain ceramides. Sphingomyelinase expression, and levels of sphingomyelin and diacylglycerol were unchanged. But surprisingly phospholipids and acyl CoAs contained increased saturated long chain fatty acids. hSptlc2 KO mice had decreased fractional shortening and thinning of the cardiac wall. While the genes regulating glucose and fatty acid metabolism were not changed, expression of cardiac failure markers and the genes involved in the formation of extracellular matrices were up-regulated in hSptlc2 KO hearts. In addition, ER-stress markers were up-regulated leading to increased apoptosis. These results suggest that Sptlc2-mediated de novo ceramide synthesis is an essential source of C18:0 and very long chain, but not of shorter chain, ceramides in the heart. Changes in heart lipids other than ceramide levels lead to cardiac toxicity.     

Ceramide selectively displaces cholesterol from ordered lipid domains (rafts): implications for lipid raft structure and function

Ceramide is a membrane lipid involved in a number of crucial biological processes. Recent evidence suggests that ceramide is likely to reside and function within lipid rafts; ordered sphingolipid and cholesterol-rich lipid domains believed to exist within many eukaryotic cell membranes. Using lipid vesicles containing co-existing raft domains and disordered fluid domains, we find that natural and saturated synthetic ceramides displace sterols from rafts. Other raft lipids remain raft-associated in the presence of ceramide, showing displacement is relatively specific for sterols. Like cholesterol-containing rafts, ceramide-rich "rafts" remain in a highly ordered state. Comparison of the sterol-displacing abilities of natural ceramides with those of saturated diglycerides and an unsaturated ceramide demonstrates that tight lipid packing is critical for sterol displacement by ceramide. Based on these results, and the fact that cholesterol and ceramides both have small polar headgroups, we propose that ceramides and cholesterol compete for association with rafts because of a limited capacity of raft lipids with large headgroups to accommodate small headgroup lipids in a manner that prevents unfavorable contact between the hydrocarbon groups of the small headgroup lipids and the surrounding aqueous environment. Minimizing the exposure of cholesterol and ceramide to water may be a strong driving force for the association of other molecules with rafts. Furthermore, displacement of sterol from rafts by ceramide is very likely to have marked effects upon raft structure and function, altering liquid ordered properties as well as molecular composition. In this regard, certain previously observed physiological processes may be a result of displacement. In particular, a direct connection to the previously observed sphingomyelinase-induced displacement of cholesterol from plasma membranes in cells is proposed.     

Regulation of mitochondrial ceramide distribution by members of the BCL-2 family

Apoptosis is an intricately regulated cellular process that proceeds through different cell type- and signal-dependent pathways. In the mitochondrial apoptotic program, mitochondrial outer membrane permeabilization by BCL-2 proteins leads to the release of apoptogenic factors, caspase activation, and cell death. In addition to protein components of the mitochondrial apoptotic machinery, an interesting role for lipids and lipid metabolism in BCL-2 family-regulated apoptosis is also emerging. We used a comparative lipidomics approach to uncover alterations in lipid profile in the absence of the proapoptotic proteins BAX and BAK in mouse embryonic fibroblasts (MEFs). We detected over 1,000 ions in these experiments and found changes in an ion with an m/z of 534.49. Structural elucidation of this ion through tandem mass spectrometry revealed that this molecule is a ceramide with a 16-carbon N-acyl chain and sphingadiene backbone (d18:2/16:0 ceramide). Targeted LC/MS analysis revealed elevated levels of additional sphingadiene-containing ceramides (d18:2-Cers) in BAX, BAK-double knockout MEFs. Elevated d18:2-Cers are also found in immortalized baby mouse kidney epithelial cells lacking BAX and BAK. These results support the existence of a distinct biochemical pathway for regulating ceramides with different backbone structures and suggest that sphingadiene-containing ceramides may have functions that are distinct from the more common sphingosine-containing species.     

Ceramide biosynthesis in keratinocyte and its role in skin function

The enucleate layer of the epidermis, i.e. the stratum corneum, is responsible for certain critical protective functions, such as epidermal permeability barrier function. Within the epidermal membrane lamella component, ceramides are the dominant lipid class by weight (over 50%) and exhibit the greatest molecular heterogeneity in terms of sphingoid base and fatty acid composition. It is now evermore important to understand how ceramide production and functions are controlled in the epidermis, since decreased epidermal ceramide content has been linked to water loss and barrier dysfunction. During the past several years, critical enzymes in ceramide biosynthesis have been identified, including ceramide synthases (CerS) and ceramide hydroxylase/desaturase. In this review, we describe the molecular heterogeneity of ceramides synthesized in the epidermis and their possible roles in epidermal permeability barrier functions. We also describe recent studies that identified the family of CerS (CerS1–CerS6) in mammals. We further focus on the roles of specific isoforms of these enzymes in synthesizing the epidermal ceramides, especially in relation to chain-length specificity. In addition, we provide experimental information, including our recent findings, as to how applying ceramide or ceramide-containing substances to skin, orally or directly, can benefit skin health.     

Ceramide and mitochondria in ischemic brain injury

Sphingolipids are essential structural components of cellular membranes, playing prominent roles in signal transduction that governs cell proliferation, differentiation and apoptosis. Ceramides, a family of distinct molecular species characterized by various acyl chains, are synthesized de novo at the cytosolic side of the endoplasmic reticulum serving as precursors for the biosynthesis of sphingolipids in the Golgi. Recently, mitochondria emerged as an important intracellular compartment of sphingolipid metabolism. Thus, several sphingolipid-metabolizing enzymes were found to be associated with mitochondria, including neutral ceramidase, novel neutral sphingomyelinase, and (dihydro) ceramide synthase, an important ceramide-generating enzyme in de novo ceramide synthesis and recycling pathway. Mitochondrial dysfunction appears to be essential in tissue damage after brain ischemia/reperfusion (IR). Mitochondria are known to be involved in both the necrosis and apoptosis detected in animal models of ischemic stroke, and treatments that ameliorate tissue infarction were associated with better recovery of mitochondrial function. Although mitochondrial injury in stroke has been extensively studied and key mitochondrial functions affected by IR are mainly characterized, the nature of the molecule that causes loss of mitochondrial integrity and function remains obscure. Emerging data indicate a deregulation of ceramide metabolism in mitochondria damaged by IR suggesting that ceramides could play critical roles in cerebral IR-induced mitochondrial damage. This review will examine the experimental evidence supporting the key role of ceramides in mitochondrial dysfunction in cerebral IR and highlight potential targets for development of novel therapeutic approaches for stroke treatment.     

Role of sphingosine kinase localization in sphingolipid signaling

The sphingosine kinases, SK1 and SK2, produce the potent signaling lipid sphingosine-1-phosphate (S1P). These enzymes have garnered increasing interest for their roles in tumorigenesis, inflammation, vascular diseases, and immunity, as well as other functions. The sphingosine kinases are considered signaling enzymes by producing S1P, and their activity is acutely regulated by a variety of agonists. However, these enzymes are also key players in the control of sphingolipid metabolism. A variety of sphingolipids, such as sphingosine and the ceramides, are potent signaling molecules in their own right. The role of sphingosine kinases in regulating sphingolipid metabolism is potentially a critical aspect of their signaling function. A central aspect of signaling lipids is that their hydrophobic nature constrains them to membranes. Most enzymes of sphingolipid metabolism, including the enzymes that degrade S1P, are membrane enzymes. Therefore the localization of the sphingosine kinases and S1P is likely to be important in S1P signaling. Sphingosine kinase localization affects sphingolipid signaling in several ways. Translocation of SK1 to the plasma membrane promotes extracellular secretion of S1P. SK1 and SK2 localization to specific sites appears to direct S1P to intracellular protein effectors. SK localization also determines the access of these enzymes to their substrates. This may be an important mechanism for the regulation of ceramide biosynthesis by diverting dihydrosphingosine, a precursor in the ceramide biosynthetic pathway, from the de novo production of ceramide.     

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.     

Downregulation of neutral ceramidase by gemcitabine: Implications for cell cycle regulation

Gemcitabine (GMZ) is a chemotherapeutic agent with well established effects on cell growth arrest and apoptosis. In this study, we investigated the potential roles of bioactive sphingolipids in mediating the growth suppressing effects of GMZ on a polyoma middle T transformed murine endothelial cell line. After 12-hour GMZ (0.6 μM) treatment, cell growth was arrested at the G₀/G₁ phase as detected by flow cytometric cell cycle analysis and MTT cell viability analysis, and this was accompanied by dephosphorylation of the retinoblastoma protein (Rb). Furthermore, GMZ treatment resulted in increased levels of specifically the very long chain ceramides as determined by mass spectrometry. Mechanistically, GMZ did not appear to affect the activities of many enzymes of ceramide metabolism; however, GMZ caused a selective reduction in the protein levels of neutral ceramidase (NCDase), as indicated by Western blot analysis, with a concomitant decrease in NCDase activity. The significance of NCDase loss on cell cycle regulation was investigated by specific knockdown of the enzyme using small interfering RNA (siRNA). Interestingly, NCDase siRNA transfection was sufficient to induce a cell cycle arrest at G₀/G₁ and an increase in total ceramide levels, with significant elevation in very long chain ceramides (C_24:1 and C_24:0). NCDase siRNA also induced Rb dephosphorylation. These data provide evidence for a novel mechanism of action for GMZ and highlight downregulation of NCDase as a critical step in GMZ-mediated ceramide elevation and cell cycle arrest.     

Exploring the link between ceramide and ionizing radiation

The aim of radiotherapy is to eradicate cancer cells with ionizing radiation; tumor cell death following irradiation can be induced by several signaling pathways, most of which are triggered as a consequence of DNA damage, the primary and major relevant cell response to radiation. Several lines of evidence demonstrated that ceramide, a crucial sensor and/or effector of different signalling pathways promoting cell cycle arrest, death and differentiation, is directly involved in the molecular mechanisms underlying cellular response to irradiation. Most of the studies strongly support a direct relationship between ceramide accumulation and radiation-induced cell death, mainly apoptosis; for this reason, defining the contribution of the multiple metabolic pathways leading to ceramide formation and the causes of its dysregulated metabolism represent the main goal in order to elucidate the ceramide-mediated signaling in radiotherapy. In this review, we summarize the current knowledge concerning the different routes leading to ceramide accumulation in radiation-induced cell response with particular regard to the role of the enzymes involved in both ceramide neogenesis and catabolism. Emphasis is placed on sphingolipid breakdown as mechanism of ceramide generation activated following cell irradiation; the functional relevance of this pathway, and the role of glycosphingolipid glycohydrolases as direct targets of ionizing radiation are also discussed. These new findings add a further attractive point of investigation to better define the complex interplay between sphingolipid metabolism and radiation therapy.     

Accumulation of specific ceramides in ischemic/reperfused rat heart; effect of ischemic preconditioning.

Ceramide signalling has been implicated in the mechanism of myocardial ischemia/reperfusion injury (IR). This study tested the hypothesis that ceramides containing a specific amino-linked acyl residue mediate the injury, and that ischemic preconditioning (IPC) affords myocardial protection because it prevents increased ceramide accumulation in IR myocardium. Perfused rat hearts were subjected either to the sham perfusion or to 30 min global ischemia, 30 min ischemia/30 min reperfusion (IR) or were preconditioned prior to the standard IR. The ventricles were harvested for biochemical assay that involved transmethylation of ceramide amino-linked acyl residues, and gas liquid chromatography measurement of acyl methyl esters. Fourteen ceramides containing myrystic, palmitic, palmitoleic, stearic, oleic, linoleic, linolenic, arachidic, arachidonic, eicosapentaenoic, behenic, docosapentaenoic, docosahexaenoic or nervonic acid were identified in the myocardium of rats. The total basal ceramide concentration in the myocardium was 135 nmol/g tissue, and it was increased by 14.1% and 48.4% in the ischemia and IR group, respectively. However, in fact, IR increased the accumulation of only 7 out of 14 ceramides identified in the heart (i.e., those containing palmitic, stearic, oleic, linoleic, and arachidonic acid), and the relative magnitude of these increases varied between the particular ceramides and was independent from their basal tissue concentration. IPC improved postischemic hemodynamic recovery and partially prevented the reperfusion-induced increases in these 7 ceramides, while the other ceramides were unaffected by IPC. These results support the role of the specific ceramide signalling in the mechanism of myocardial IR injury. We speculate that by preventing tissue accumulation of certain ceramides, IPC attenuates this signalling, that adds to the mechanism of myocardial protection afforded by IPC.     

Lipid remodeling leads to the introduction and exchange of defined ceramides on GPI proteins in the ER and Golgi of Saccharomyces cerevisiae.

Previous experiments with Saccharomyces cerevisiae had suggested that diacylglycerol-containing glycosylphosphatidylinositols (GPIs) are added to newly synthesized proteins in the endoplasmic reticulum (ER) and that ceramides subsequently are incorporated into GPI proteins by lipid remodeling. Here we prove this hypothesis by labeling yeast cells with [3H]dihydrosphingosine ([3H]DHS) and showing that this tracer is incorporated into many GPI proteins even when protein synthesis and, hence, anchor addition, is blocked by cycloheximide. [3H]DHS incorporation is greatly enhanced if endogenous synthesis of DHS is inhibited by myriocin. Labeled GPI anchors contain three types of ceramides which, based on previous and present results, are identified as DHS-C26:0, phytosphingosine-C26:0 and phytosphingosine-C26:0-OH, the latter being found only on proteins which have reached the Golgi. Lipid remodeling can occur both in the ER and in a later secretory compartment. In addition, ceramide is incorporated into GPI proteins a long time after their initial synthesis by a process in which one ceramide gets replaced by another ceramide. Remodeling outside the ER requires vesicular flow from the ER to the Golgi, possibly to supply the remodeling enzymes with ceramides.     

Stinging Nettle (Urtica dioica L.) Attenuates FFA Induced Ceramide Accumulation in 3T3-L1 Adipocytes in an Adiponectin Dependent Manner

Objective

Excess dietary lipids result in the accumulation of lipid metabolites including ceramides that can attenuate insulin signaling. There is evidence that a botanical extract of Urtica dioica L. (stinging nettle) improves insulin action, yet the precise mechanism(s) are not known. Hence, we examined the effects of Urtica dioica L. (UT) on adipocytes.

Research Design

We investigated the effects of an ethanolic extract of UT on free fatty acid (palmitic acid) induced inhibition of insulin-stimulated Akt serine phosphorylation and modulation of ceramidase expression in 3T3-L1 adipocytes. Adipocytes were exposed to excess FFAs in the presence or absence of UT. Effects on adiponectin expression, ceramidase expression, ceramidase activity, ceramide accumulation and insulin signaling were determined.

Results

As expected, FFAs reduced adiponectin expression and increased the expression of ceramidase enzymes but not their activity. FFA also induced the accumulation of ceramides and reduced insulin-stimulated phosphorylation of Akt in adipocytes. The effects of FFA were partially reversed by UT. UT enhanced adiponectin expression and ceramidase activity in the presence of excess FFAs. UT abated ceramide accumulation and increased insulin sensitivity via enhanced Akt phosphorylation. A siRNA knockdown of adiponectin expression prevented UT from exerting positive effects on ceramidase activity but not Akt phosphorylation.

Conclusions

In adipocytes, the ability of UT to antagonize the negative effects of FFA by modulating ceramidase activity and ceramide accumulation is dependent on the presence of adiponectin. However, the ability of UT to enhance Akt phosphorylation is independent of adiponectin expression. These studies demonstrate direct effects of UT on adipocytes and suggest this botanical extract is metabolically beneficial.     

Altered hepatic lipid metabolism in C57BL/6 mice fed alcohol: a targeted lipidomic and gene expression study

Chronic alcohol consumption is associated with fatty liver disease in mammals. The object of this study was to gain an understanding of dysregulated lipid metabolism in alcohol-fed C57BL/6 mice using a targeted lipidomic approach. Liquid chromatography tandem mass spectrometry was used to analyze several lipid classes, including free fatty acids, fatty acyl-CoAs, fatty acid ethyl esters, sphingolipids, ceramides, and endocannabinoids, in plasma and liver samples from control and alcohol-fed mice. The interpretation of lipidomic data was augmented by gene expression analyses for important metabolic enzymes in the lipid pathways studied. Alcohol feeding was associated with i) increased hepatic free fatty acid levels and decreased fatty acyl-CoA levels associated with decreased mitochondrial fatty acid oxidation and decreased fatty acyl-CoA synthesis, respectively; ii) increased hepatic ceramide levels associated with higher levels of the precursor molecules sphingosine and sphinganine; and iii) increased hepatic levels of the endocannabinoid anandamide associated with decreased expression of its catabolic enzyme fatty acid amide hydrolase. The unique combination of lipidomic and gene expression analyses allows for a better mechanistic understanding of dysregulated lipid metabolism in the development of alcoholic fatty liver disease.     

Effect of exercise duration on the key pathways of ceramide metabolism in rat skeletal muscles

Ceramide is the key compound on crossroads of sphingolipid metabolism. The content and composition of ceramides in skeletal muscles have been shown to be affected by prolonged exercise. The aim of this study was to examine the effect of exercise on the activity of key enzymes of ceramide metabolism in skeletal muscles. The experiments were carried out on male Wistar rats (200-250 g) divided into four groups: sedentary, exercised for 30 min, 90 min, and until exhaustion. The activity of serine palmitoyltransferase (SPT), neutral and acid sphingomyelinase (nSMase and aSMase), neutral and alkaline ceramidases (nCDase and alCDase) and the content of ceramide, sphingosine, sphinganine and sphingosine-1-phosphate were determined in three types of muscle. We have found that the activity and expression of SPT increase gradually in each muscle with duration of exercise. These changes were followed by elevation in the content of sphinganine. These data indicate that exercise increases de novo synthesis of ceramide. The aSMase activity gradually decreased with duration of exercise in each type of muscle. After exhaustive exercise the activity of both isoforms of ceramidase were reduced in each muscle. The ceramide level depends both on duration of exercise and muscle type. The ceramide level in the soleus and white gastrocnemius decreased after 30 min of running. After exhaustive exercise it was elevated in the soleus and red gastrocnemius. It is concluded that exercise strongly affects the activity of key enzymes involved in ceramide metabolism and in consequence the level of sphingolipid intermediates in skeletal muscles.     

Ceramide channels increase the permeability of the mitochondrial outer membrane to small proteins

Ceramides are known to play a major regulatory role in apoptosis by inducing cytochrome c release from mitochondria. We have previously reported that C(2)- and C(16)-ceramide, but not dihydroceramide, form large channels in planar membranes (Siskind, L. J., and Colombini, M. (2001) J. Biol. Chem. 275, 38640-38644). Here we show that ceramides do not trigger a cytochrome c secretion or release mechanism, but simply raise the permeability of the mitochondrial outer membrane, via ceramide channel formation, to include small proteins. Exogenously added reduced cytochrome c was able to freely permeate the mitochondrial outer membrane with entry to and exit from the intermembrane space facilitated by ceramides in a dose- and time-dependent manner. The permeability pathways were eliminated upon removal of C(2)-ceramide by bovine serum albumin, thus ruling out a detergent-like effect of C(2)-ceramide on membranes. Ceramide channels were not specific to cytochrome c, as ceramides induced release of adenylate kinase, but not fumerase from isolated mitochondria, showing some specificity of these channels for the outer mitochondrial membrane. SDS-PAGE results show that ceramides allow release of intermembrane space proteins with a molecular weight cut-off of about 60,000. These results indicate that the ceramide-induced membrane permeability increases in isolated mitochondria are via ceramide channel formation and not a release mechanism, as the channels that allow cytochrome c to freely permeate are reversible, and are not specific to cytochrome c.     

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.     

Alkaline ceramidase 3 deficiency aggravates colitis and colitis-associated tumorigenesis in mice by hyperactivating the innate immune system

Increasing studies suggest that ceramides differing in acyl chain length and/or degree of unsaturation have distinct roles in mediating biological responses. However, still much remains unclear about regulation and role of distinct ceramide species in the immune response. Here, we demonstrate that alkaline ceramidase 3 (Acer3) mediates the immune response by regulating the levels of C_18:1-ceramide in cells of the innate immune system and that Acer3 deficiency aggravates colitis in a murine model by augmenting the expression of pro-inflammatory cytokines in myeloid and colonic epithelial cells (CECs). According to the NCBI Gene Expression Omnibus (GEO) database, ACER3 is downregulated in immune cells in response to lipopolysaccharides (LPS), a potent inducer of the innate immune response. Consistent with these data, we demonstrated that LPS downregulated both Acer3 mRNA levels and its enzymatic activity while elevating C_18:1-ceramide, a substrate of Acer3, in murine immune cells or CECs. Knocking out Acer3 enhanced the elevation of C_18:1-ceramide and the expression of pro-inflammatory cytokines in immune cells and CECs in response to LPS challenge. Similar to Acer3 knockout, treatment with C_18:1-ceramide, but not C_18:0-ceramide, potentiated LPS-induced expression of pro-inflammatory cytokines in immune cells. In the mouse model of dextran sulfate sodium-induced colitis, Acer3 deficiency augmented colitis-associated elevation of colonic C_18:1-ceramide and pro-inflammatory cytokines. Acer3 deficiency aggravated diarrhea, rectal bleeding, weight loss and mortality. Pathological analyses revealed that Acer3 deficiency augmented colonic shortening, immune cell infiltration, colonic epithelial damage and systemic inflammation. Acer3 deficiency also aggravated colonic dysplasia in a mouse model of colitis-associated colorectal cancer. Taken together, these results suggest that Acer3 has an important anti-inflammatory role by suppressing cellular or tissue C_18:1-ceramide, a potent pro-inflammatory bioactive lipid and that dysregulation of ACER3 and C_18:1-ceramide may contribute to the pathogenesis of inflammatory diseases including cancer.Ceramides are the central lipid in the metabolic network of sphingolipids, and are generated through the de novo, catabolic and salvage pathways.¹ In the de novo pathway, ceramides are synthesized through multiple steps catalyzed sequentially by serine palmitoyltransferase (SPT), keto-dihydrosphingosine reductase, (dihydro)ceramide synthases (CerSs) and dihydroceramide desaturases. In the catabolic pathways, ceramides are derived from the hydrolysis of sphingomyelins by sphingomyelinases (SMases) or the hydrolysis of glycosphingolipids. In the salvage pathway, ceramides are synthesized from sphingosine (SPH) and fatty acyl-CoA by CerSs. As CerSs (CerS1-6) have distinct specificity toward acyl-CoA chain length and degree of unsaturation, ceramides with various acyl-chains are found in mammalian cells. Upon generation, ceramides can be hydrolyzed by five ceramidases encoded by five distinct genes (ASAH1, ASAH2, ACER1, ACER2 and ACER3). These ceramidases vary in pH optimum for catalytic activity, tissue distribution, cellular localization and substrate specificity,² allowing for regulation of specific ceramides in a cell- or tissue-specific manner.Recent studies have implicated ceramides in regulating the innate immune response. Sakata et al.³ demonstrated that lipopolysaccharides (LPS), a potent inducer of the innate immune response, increases C₁₆-ceramide by activating acid SMase and that inhibition of SMase attenuates LPS-induced production of pro-inflammatory cytokines in THP-1 macrophages. Andreyev et al.⁴ found that ceramides are increased by Toll-like receptor 4 (TLR4)-specific LPS in RAW 264.7 macrophages. Schilling et al.⁵ revealed that LPS and palmitic acid synergistically increase C₁₆-ceramide in primary mouse peritoneal macrophages (PMs) by activating de novo biosynthesis of ceramides and that inhibiting the C₁₆-ceramide increase attenuates LPS-induced production of TNF-α and IL-1β in PMs. A recent study found that LPS increases ceramides in Raw 264.7 macrophages through nuclear factor kappa B (NF-κB)-dependent upregulation of SPT long chain base subunit 2 Sptlc2, a regulator of SPT.⁶ These results suggest that ceramides mediate the immune response in part by enhancing the production of pro-inflammatory cytokines in innate immune cells.Emerging evidence suggests that dysregulation in the innate immune response in inflammatory bowel disease (IBD) contributes to the pathogenesis of the disease.⁷ Consistent with the role of ceramides in potentiating the innate immune response, several studies found that ceramides may have a role in the pathogenesis of IBD. Sakata et al.³ demonstrated that blocking the generation of ceramides with the SMase inhibitor hinders mouse colitis. Fischbeck et al.⁸ showed that increasing ceramides in the gut by supplying mice with dietary sphingomyelins, a precursor of ceramides, aggravates mouse colitis. These results suggest that increased levels of ceramides may contribute to the pathogenesis of IBD.Although the role of ceramides and their generating enzymes in the innate immune response have been well studied, much remains unclear about the role of ceramidases involved in the catabolism of ceramides in this biological response. In this study, we investigated the role of alkaline ceramidase 3 (ACER3)/Acer3 and its substrates in immune response. We demonstrated that Acer3 is downregulated, whereas its substrate, C_18:1-ceramide, is upregulated in murine immune cells and colonic epithelial cells (CECs) during the innate immune response to LPS. Using Acer3 null mice (Acer3^−/−) and their wild-type (Acer3^+/+) littermates, we further discovered that the inverse regulation of Acer3 and C_18:1-ceramide potentiates LPS-induced production of pro-inflammatory cytokines in innate immune cells. More importantly, we found that Acer3 deficiency aggravates dextran sulfate sodium (DSS)-induced colitis and colitis-associated colorectal cancer (CAC) in a murine model. These findings indicate that Acer3/ACER3 and C_18:1-ceramide are novel modulators in the innate immune response and that their dysregulation may contribute to the pathogenesis of inflammatory diseases.