Ceramide induces interleukin 6 gene expression in human fibroblasts.

We previously reported that ceramide, the immediate product of sphingomyelin hydrolysis, increases in response to interleukin (IL)-1 beta and plays a role in modulating IL-1 beta-mediated prostaglandin E2 production and cyclooxygenase gene expression in human fibroblasts (Ballou, L. R., C. P. Chao, M. A. Holness, S. C. Barker, and R. Raghow. 1992. J. Biol. Chem. 267:20044-20050). Here we describe the effects of ceramide in another IL-1 beta-mediated process in these cells, the induction of IL-6 production. We found that submicromolar concentrations of C2-ceramide induced IL-6 gene expression and protein production as effectively as IL-1 beta. Both D-erythro-C2-ceramide (a cell-permeable analogue of natural ceramide) and D-threo-C2-ceramide were potent inducers of IL-6 production, while neither L isomer of ceramide was effective. Compared with IL-1 beta-induced IL-6 production, cells treated with ceramide or exogenous sphingomyelinase induced 82 and 50% of maximal IL-1 beta-induced IL-6 levels by 6 h, respectively; by 24 h all three treatments induced similar levels of IL-6 production. Ceramide-induced IL-6 messenger RNA could be detected within 1 h of treatment and reached maximal levels by 24 h. These findings suggest that ceramide may play a role in the regulation of IL-6 gene expression.

Cytokine response modifier A (CrmA) inhibits ceramide formation in response to tumor necrosis factor (TNF)-alpha: CrmA and Bcl-2 target distinct components in the apoptotic pathway.

Proteases are now firmly established as major regulators of the "execution" phase of apoptosis. Here, we examine the role of proteases and their relationship to ceramide, a proposed mediator of apoptosis, in the tumor necrosis factor-alpha (TNF-alpha)-induced pathway of cell death. Ceramide induced activation of prICE, the protease that cleaves the death substrate poly(ADP-ribose) polymerase. Bcl-2 inhibited ceramide-induced death, but not ceramide generation. In contrast, Cytokine response modifier A (CrmA), a potent inhibitor of Interleukin-1 beta converting enzyme and related proteases, inhibited ceramide generation and prevented TNF-alpha-induced death. Exogenous ceramide could overcome the CrmA block to cell death, but not the Bcl-2 block. CrmA, however, did not inhibit the activation of nuclear factor (NF)-kappa B by TNF-alpha, demonstrating that other signaling functions of TNF-alpha remain intact and that ceramide does not play a role in the activation of NF-kappa B. These studies support a distinct role for proteases in the signaling/activation phase of apoptosis acting upstream of ceramide formation.

Ceramide in the eukaryotic stress response.

Several extracellular agents and stress stimuli, such as tumour necrosis factor alpha, chemotherapeutic agents and heat, cause ceramide accumulation. They do this by regulating enzymes involved in its metabolism. Ceramide modulates a number of biochemical and cellular responses to stress, including apoptosis, cell-cycle arrest and cell senescence.

Inhibition of caspases inhibits the release of apoptotic bodies: Bcl-2 inhibits the initiation of formation of apoptotic bodies in chemotherapeutic agent-induced apoptosis.

During apoptosis, the cell actively dismantles itself and reduces cell size by the formation and pinching off of portions of cytoplasm and nucleus as "apoptotic bodies." We have combined our previously established quantitative assay relating the amount of release of [3H]-membrane lipid to the degree of apoptosis with electron microscopy (EM) at a series of timepoints to study apoptosis of lymphoid cells exposed to vincristine or etoposide. We find that the [3H]-membrane lipid release assay correlates well with EM studies showing the formation and release of apoptotic bodies and cell death, and both processes are regulated in parallel by inducers or inhibitors of apoptosis. Overexpression of Bcl-2 or inhibition of caspases by DEVD inhibited equally well the activation of caspases as indicated by PARP cleavage. They also inhibited [3H]-membrane lipid release and release of apoptotic bodies. EM showed that cells overexpressing Bcl-2 displayed near-normal morphology and viability in response to vincristine or etoposide. In contrast, DEVD did not prevent cell death. Although DEVD inhibited the chromatin condensation, PARP cleavage, release of apoptotic bodies, and release of labeled lipid, DEVD-treated cells showed accumulation of heterogeneous vesicles trapped in the condensed cytoplasm. These results suggest that inhibition of caspases arrested the maturation and release of apoptotic bodies. Our results also imply that Bcl-2 regulates processes in addition to caspase activation.

Tissue and cellular distribution of the extended family of protein kinase C isoenzymes.

Polyclonal isoenzyme-specific antisera were developed against four calcium-independent protein kinase C (PKC) isoenzymes (delta, epsilon, epsilon', and zeta) as well as the calcium-dependent isoforms (alpha, beta I, beta II, and gamma). These antisera showed high specificities, high titers, and high binding affinities (3-370 nM) for the peptide antigens to which they were raised. Each antiserum detected a species of the predicted molecular weight by Western blot that could be blocked with the immunizing peptide. PKC was sequentially purified from rat brain, and the calcium-dependent forms were finally resolved by hydroxyapatite chromatography. Peak I reacted exclusively with antisera to PKC gamma, peak II with PKC beta I and -beta II, and peak III with PKC alpha. These same fractions, however, were devoid of immunoreactivity for the calcium-independent isoenzymes. The PKC isoenzymes demonstrated a distinctive tissue distribution when evaluated by Western blot and immunocytochemistry. PCK delta was present in brain, heart, spleen, lung, liver, ovary, pancreas, and adrenal tissues. PKC epsilon was present in brain, kidney, and pancreas, whereas PKC epsilon' was present predominantly in brain. PKC zeta was present in most tissues, particularly the lung, brain, and liver. Both PKC delta and PKC zeta showed some heterogeneity of size among the different tissues. PKC alpha was present in all organs and tissues examined. PKC beta I and -beta II were present in greatest amount in brain and spleen. Although the brain contained the most PKC gamma immunoreactivity, some immunostaining was also seen in adrenal tissue. These studies provide the first evidence of selective organ and tissue distributions of the calcium-independent PKC isoenzymes.

Functions of ceramide in coordinating cellular responses to stress.

Sphingolipid metabolites participate in key events of signal transduction and cell regulation. In the sphingomyelin cycle, a number of extracellular agents and insults (such as tumor necrosis factor, Fas ligands, and chemotherapeutic agents) cause the activation of sphingomyelinases, which act on membrane sphingomyelin and release ceramide. Multiple experimental approaches suggest an important role for ceramide in regulating such diverse responses as cell cycle arrest, apoptosis, and cell senescence. In vitro, ceramide activates a serine-threonine protein phosphatase, and in cells it regulates protein phosphorylation as well as multiple downstream targets [such as interleukin converting enzyme (ICE)-like proteases, stress-activated protein kinases, and the retinoblastoma gene product] that mediate its distinct cellular effects. This spectrum of inducers of ceramide accumulation and the nature of ceramide-mediated responses suggest that ceramide is a key component of intracellular stress response pathways.

Lysosphingolipids inhibit protein kinase C: implications for the sphingolipidoses.

Lysosphingolipids potently and reversibly inhibited protein kinase C activity and binding of phorbol dibutyrate in vitro and in human platelets. As with activation of protein kinase C by phosphatidylserine and sn-1,2-diacylglycerol, inhibition was subject to surface dilution. Accordingly, inhibition in mixed micelle assays was dependent on the molar percentage of lysosphingolipids rather than the bulk concentration. Lysosphingolipids inhibited protein kinase C activity at molar percentages similar to those required for activation by phosphatidylserine and sn-1,2-diacylglycerol. Since lysosphingolipids accumulate in Krabbe's disease, Gaucher's disease, and other sphingolipidoses, the hypothesis that lysosphingolipid inhibition of protein kinase C represents the missing functional link between the accumulation of sphingolipids and the pathogenesis of these disorders appears to unify existing data. The accumulation of lysosphingolipids would cause progressive dysfunction of signal transduction mechanisms vital for neural transmission, differentiation, development, and proliferation and would eventually lead to cell death.

Functions of sphingolipids and sphingolipid breakdown products in cellular regulation.

The discovery that breakdown products of cellular sphingolipids are biologically active has generated interest in the role of these molecules in cell physiology and pathology. Sphingolipid breakdown products, sphingosine and lysosphingolipids, inhibit protein kinase C, a pivotal enzyme in cell regulation and signal transduction. Sphingolipids and lysosphingolipids affect significant cellular responses and exhibit antitumor promoter activities in various mammalian cells. These molecules may function as endogenous modulators of cell function and possibly as second messengers.

Programmed cell death induced by ceramide.

Sphingomyelin hydrolysis and ceramide generation have been implicated in a signal transduction pathway that mediates the effects of tumor necrosis factor-alpha (TNF-alpha) and other agents on cell growth and differentiation. In many leukemic cells, TNF-alpha causes DNA fragmentation, which leads to programmed cell death (apoptosis). C2-ceramide (0.6 to 5 microM), a synthetic cell-permeable ceramide analog, induced internucleosomal DNA fragmentation, which was inhibited by zinc ion. Other amphiphilic lipids failed to induce apoptosis. The closely related C2-dihydroceramide was also ineffective, which suggests a critical role for the sphingolipid double bond. The effects of C2-ceramide on DNA fragmentation were prevented by the protein kinase C activator phorbol 12-myristate 13-acetate, which suggests the existence of two opposing intracellular pathways in the regulation of apoptosis.

Activation of the sphingomyelin cycle through the low-affinity neurotrophin receptor.

The role of the low-affinity neurotrophin receptor (p75NTR) in signal transduction is undefined. Nerve growth factor can activate the sphingomyelin cycle, generating the putative-lipid second messenger ceramide. In T9 glioma cells, addition of a cell-permeable ceramide analog mimicked the effects of nerve growth factor on cell growth inhibition and process formation. This signaling pathway appears to be mediated by p75NTR in T9 cells and NIH 3T3 cells overexpressing p75NTR. Expression of an epidermal growth factor receptor-p75NTR chimera in T9 cells imparted to epidermal growth factor the ability to activate the sphingomyelin cycle. These data demonstrate that p75NTR is capable of signaling independently of the trk neurotrophin receptor (p140trk) and that ceramide may be a mediator in neurotrophin biology.

Ceramide: an intracellular signal for apoptosis.

Recent investigation of the roles of sphingolipids in signal transduction and cell regulation is shedding a new light on the mechanisms of growth suppression and apoptosis. A sphingomyelin cycle has been identified whereby the action of certain extracellular agents (such as tumor necrosis factor alpha) results in activation of a sphingomyelinase, which cleaves membrane sphingomyelin, to generate cellular ceramide. Ceramide, in turn, has emerged as a candidate intracellular mediator for the action of these extracellular agents, and has multiple cellular and biochemical targets. In particular, ceramide is a potent and specific suppressor of cell growth and an inducer of apoptosis. Further studies on this signal transduction pathway should provide new understanding of the physiological functions of ceramide and promise significant insight into a novel biochemical pathway regulating apoptosis.

Sphingolipid-binding proteins.

Emerging information on sphingolipid metabolism and signaling is leading to a better understanding of cellular processes such as apoptosis, cancer, cell survival and aging. In this review, we discuss the involvement of sphingolipids in these processes and focus on underlying mechanisms based on sphingolipid:protein interactions. Due to the inherent difficulty of studying lipids, we discuss techniques that are useful in the elucidation of these interactions. We classify sphingolipid-binding proteins into four main classes: receptor, effector, enzyme, and transporter. Known structures of sphingolipid-binding proteins are surveyed, and sphingolipid-binding characteristics are described, acknowledging the limitations that there are presently insufficient protein:sphingolipid complexes for more definitive conclusions on this topic. Finally we summarize relevant literature to better inform the reader about sphingolipid:protein interactions.

Cytosolic Ca2+ and protein kinase Calpha couple cellular metabolism to membrane K+ permeability in a human biliary cell line.

Cholangiocytes represent an important target of injury during the ischemia and metabolic stress that accompanies liver preservation. Since K+ efflux serves to minimize injury during ATP depletion in certain other cell types, the purpose of these studies was to evaluate the effects of ATP depletion on plasma membrane K+ permeability of Mz-ChA-1 cells, a model human biliary cell line. Cells were exposed to dinitrophenol (50 microM) and 2-deoxyglucose (10 mM) as the standard model of metabolic injury. Whole-cell and single K+ channel currents were measured using patch clamp techniques; and intracellular [Ca2+] ([Ca2+]i) was estimated by calcium green-1 fluorescence. Metabolic stress increased [Ca2+]i, and stimulated translocation of the alpha isoform of protein kinase C (PKCalpha) from cytosolic to particulate cell fractions. The same maneuver increased membrane K+ permeability 40-70-fold as detected by (a) activation of K+selective whole cell currents of 2,176+/-218 pA (n = 34), and (b) opening of apamin-sensitive K+ channels with a unitary conductance of 17.0+/-0.2 pS. PKCalpha translocation and channel opening appear to be related since stress-induced K+ efflux is inhibited by chelation of cytosolic Ca2+, exposure to the PKC inhibitor chelerythrine (25 microM) and downregulation of PKC by phorbol esters. Moreover, K+ currents were activated by intracellular perfusion with recombinant PKCalpha in the absence of metabolic inhibitors. These findings indicate that in biliary cells apamin-sensitive K+ channels are functionally coupled to cell metabolism and suggest that cytosolic Ca2+ and PKCalpha are selectively involved in the response.

p53-dependent ceramide response to genotoxic stress.

Both p53 and ceramide have been implicated in the regulation of growth suppression. p53 has been proposed as the "guardian of the genome" and ceramide has been suggested as a "tumor suppressor lipid. " Both molecules appear to regulate cell cycle arrest, senescence, and apoptosis. In this study, we investigated the relationship between p53 and ceramide. We found that treatment of Molt-4 cells with low concentrations of actinomycin D or gamma-irradiation, which activate p53-dependent apoptosis, induces apoptosis only in cells expressing normal levels of p53. In these cells, p53 activation was followed by a dose- and time-dependent increase in endogenous ceramide levels which was not seen in cells lacking functional p53 and treated similarly. Similar results were seen in irradiated L929 cells whereby the p53-deficient clone was significantly more resistant to irradiation and exhibited no ceramide response. However, in p53-independent systems, such as growth suppression induced by TNF-alpha or serum deprivation, ceramide accumulated irrespective of the upregulation of p53, indicating that p53 regulates ceramide accumulation in only a subset of growth-suppressive pathways. Finally, ceramide did not increase p53 levels when used at growth-suppressive concentrations. Also, when cells lacking functional p53, either due to mutation or the expression of the E6 protein of human papilloma virus, were treated with exogenous ceramide, there was equal growth suppression, cell cycle arrest, and apoptosis as compared with cells expressing normal p53. These results indicate that p53 is unlikely to function "downstream" of ceramide. Instead, they suggest that, in situations where p53 performs a critical regulatory role, such as the response to genotoxic stress, it functions "upstream" of ceramide. These studies begin to define a relationship between these two pathways of growth inhibition.

P38 delta MAPK promotes breast cancer progression and lung metastasis by enhancing cell proliferation and cell detachment.

The protein p38 mitogen-activated protein kinase (MAPK) delta isoform (p38δ) is a poorly studied member of the MAPK family. Data analysis from The Cancer Genome Atlas database revealed that p38δ is highly expressed in all types of human breast cancers. Using a human breast cancer tissue array, we confirmed elevation in cancer tissue. The breast cancer mouse model, MMTV-PyMT (PyMT), developed breast tumors with lung metastasis; however, mice deleted in p38δ (PyMT/p38δ-/-) exhibited delayed primary tumor formation and highly reduced lung metastatic burden. At the cellular level, we demonstrate that targeting of p38δ in breast cancer cells, MCF-7 and MDA-MB-231 resulted in a reduced rate of cell proliferation. In addition, cells lacking p38δ also displayed an increased cell-matrix adhesion and reduced cell detachment. This effect on cell adhesion was molecularly supported by the regulation of the focal adhesion kinase by p38δ in the human breast cell lines. These studies define a previously unappreciated role for p38δ in breast cancer development and evolution by regulating tumor growth and altering metastatic properties. This study proposes MAPK p38δ protein as a key factor in breast cancer. Lack of p38δ resulted in reduced primary tumor size and blocked the metastatic potential to the lungs.

Distribution and dynamic changes of sphingolipids in blood in response to platelet activation.

BACKGROUND: Sphingolipids are signaling molecules in a range of biological processes. While sphingosine-1-phosphate (S1P) is thought to be abundantly stored in platelets and released upon stimulation, knowledge about the distribution and function of other sphingolipids in blood is lacking. OBJECTIVES: To analyze the sphingolipid content of blood components with special emphasis on dynamic changes in platelets. METHODS: Blood components from mice and humans were prepared by gradient centrifugation and analyzed by liquid chromatography-mass spectrometry. Additionally, murine platelets were activated in vitro and in vivo. RESULTS: Isolated non-activated platelets of mice were devoid of S1P, but instead contained dihydrosphingosine-1-phosphate (dhS1P), along with a high concentration of ceramide. Activation of platelets in vitro led to a loss of dhS1P and an increase in sphingosine, accompanied by a reduction of ceramide content. Platelet activation in vivo led to an immediate and continuous rise of dhS1P in plasma, while S1P remained stable. The sphingolipid distribution of human blood was markedly different from mice. Human platelets contained dhS1P in addition to S1P. CONCLUSIONS: Mouse platelets contain dhS1P instead of S1P. Platelet activation causes loss of dhS1P and breakdown of ceramide, implying ceramidase activation. Release of dhS1P from activated platelets might be a novel signaling pathway. Finally, the sphingolipid composition of mouse and human blood shows large differences, which must be considered when studying sphingolipid biology.

12(S)-HETE enhancement of prostate tumor cell invasion: selective role of PKC alpha.

BACKGROUND: Prostate carcinoma has become the second most fatal cancer in American men. In Dunning R3327 rat prostate adenocarcinoma cells, elevated invasiveness positively correlates with metastatic potential. However, the mechanism(s) responsible for regulation of tumor cell motility and invasion is poorly understood. We have reported that a lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], augments tumor cell metastatic potential through activation of protein kinase C (PKC). PURPOSE: We proposed to determine the effect of 12(S)-HETE on the motility and invasion of low-metastatic rat prostate AT2.1 tumor cells and the effect of 12(S)-HETE activation of specific PKC isoform(s) in these processes. METHODS: The motility of AT2.1 cells was determined by the colloidal gold phagokinetic track assay and the invasiveness measured as their ability to invade through basement membrane Matrigel-coated filters. Expression of PKC isoforms was determined by Western blotting of the whole cell lysate with isoform-specific anti-PKC antibodies. Cytosol and membrane fractions were prepared and the subcellular distribution of PKC was analyzed by Western blotting and activity assay. The effect of 12(S)-HETE on cell proliferation was examined. Data were analyzed for significance of difference with the two-sampled, two-sided Student's t test. RESULTS: 12(S)-HETE increased the motility and invasion of AT2.1 cells, and this 12(S)-HETE-increased motility and invasion were inhibited by a selective PKC inhibitor, calphostin C, as well as a Ca2 chelator, bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/tetra(acetoxy-methyl)ester. AT2.1 cells expressed the PKC isoforms alpha and delta, and 12(S)-HETE increased the membrane association of PKC alpha but not delta. Further, the motility and invasion of AT2.1 cells were increased by thymelea toxin, a selective activator of PKC alpha over PKC delta. CONCLUSION: 12(S)-HETE augments the invasiveness of AT2.1 cells via selective activation of PKC alpha. IMPLICATIONS: 12(S)-HETE modulation of PKC alpha invasiveness may be an important mechanism of action for the regulation of the invasive potential of rat prostate carcinoma cells, and the 12-lipoxygenase enzyme and/or PKC alpha may serve as key targets for the development of anti-invasive agents useful for combating the spread of prostate cancer.

Induction of apoptotic cell death and prevention of tumor growth by ceramide analogues in metastatic human colon cancer.

Dysfunction in the physiological pathways of programmed cell death may promote proliferation of malignant cells, and correction of such defects may selectively induce apoptosis in cancer cells. We measured the levels of ceramide, a candidate lipid mediator of apoptosis, in human metastatic colorectal cancer and tested in vitro and in vivo effects of various ceramide analogues in inducing apoptosis in metastatic colon cancer. Human colon cancer showed a > 50% decrease in the cellular content of ceramide when compared with normal colon mucosa. Application of ceramide analogues and ceramidase inhibitors induced rapid cell death through activation of various proapoptotic molecules, such as caspases and release of cytochrome c. Ceramidase inhibition increases the ceramide content of tumor cells, resulting in maximum activation of the apoptotic cascade. Normal liver cells were completely resistant to inhibitors of ceramidases. Treatment of nude mice with B13, the most potent ceramidase inhibitor, completely prevented tumor growth using two different aggressive human colon cancer cell lines metastatic to the liver. Therefore, B13 and related analogues of ceramide and inhibitors of ceramidases offer a promising therapeutic strategy with selective toxicity toward malignant but not normal cells. These studies also suggest that the ceramide content in cancer cells might be involved in the pathogenesis of tumor growth in vitro and in vivo.

Ceramide limits phosphatidylinositol-3-kinase C2ß-controlled cell motility in ovarian cancer: potential of ceramide as a metastasis-suppressor lipid.

Targeting cell motility, which is required for dissemination and metastasis, has therapeutic potential for ovarian cancer metastasis, and regulatory mechanisms of cell motility need to be uncovered for developing novel therapeutics. Invasive ovarian cancer cells spontaneously formed protrusions, such as lamellipodia, which are required for generating locomotive force in cell motility. Short interfering RNA screening identified class II phosphatidylinositol 3-kinase C2ß (PI3KC2ß) as the predominant isoform of PI3K involved in lamellipodia formation of ovarian cancer cells. The bioactive sphingolipid ceramide has emerged as an antitumorigenic lipid, and treatment with short-chain C6-ceramide decreased the number of ovarian cancer cells with PI3KC2ß-driven lamellipodia. Pharmacological analysis demonstrated that long-chain ceramide regenerated from C6-ceramide through the salvage/recycling pathway, at least in part, mediated the action of C6-ceramide. Mechanistically, ceramide was revealed to interact with the PIK-catalytic domain of PI3KC2ß and affect its compartmentalization, thereby suppressing PI3KC2ß activation and its driven cell motility. Ceramide treatment also suppressed cell motility promoted by epithelial growth factor, which is a prometastatic factor. To examine the role of ceramide in ovarian cancer metastasis, ceramide liposomes were employed and confirmed to suppress cell motility in vitro. Ceramide liposomes had an inhibitory effect on peritoneal metastasis in a murine xenograft model of human ovarian cancer. Metastasis of PI3KC2ß knocked-down cells was insensitive to treatment with ceramide liposomes, suggesting specific involvement of ceramide interaction with PI3KC2ß in metastasis suppression. Our study identified ceramide as a bioactive lipid that limits PI3KC2ß-governed cell motility, and ceramide is proposed to serve as a metastasis-suppressor lipid in ovarian cancer. These findings could be translated into developing ceramide-based therapy for metastatic diseases.

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 C18:1-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 C18:0-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.