Search

Emerging clinical data show that three ceramide molecules, Cer d18:1/16:0, Cer d18:1/24:1, and Cer d18:1/24:0, are biomarkers of a fatal outcome in patients with cardiovascular disease. This finding raises basic questions about their metabolic origin, their contribution to disease pathogenesis, and the utility of targeting the underlying enzymatic machinery for treatment of cardiometabolic disorders. Here, we outline the development of a potent N-acetylgalactosamine-conjugated antisense oligonucleotide engineered to silence ceramide synthase 2 specifically in hepatocytes in vivo. We demonstrate that this compound reduces the ceramide synthase 2 mRNA level and that this translates into efficient lowering of protein expression and activity as well as Cer d18:1/24:1 and Cer d18:1/24:0 levels in liver. Intriguingly, we discover that the hepatocyte-specific antisense oligonucleotide also triggers a parallel modulation of blood plasma ceramides, revealing that the biomarkers predictive of cardiovascular death are governed by ceramide biosynthesis in hepatocytes. Our work showcases a generic therapeutic framework for targeting components of the ceramide enzymatic machinery to disentangle their roles in disease causality and to explore their utility for treatment of cardiometabolic disorders. ispartof: MOLECULAR THERAPY vol:30 issue:4 pages:1661-1674 ispartof: location:United States status: published

The endoplasmic reticulum (ER) is not only important for protein synthesis and folding but is also crucial for lipid synthesis and metabolism. In the current study, we demonstrate an important role of ceramide synthases (CerS) in ER stress and NAFLD progression. Ceramide is important in sphingolipid metabolism, and its acyl chain length is determined by a family of six CerS in mammals. CerS2 generates C22-C24 ceramides, and CerS5 or CerS6 produces C16 ceramide. To gain insight into the role of CerS in NAFLD, we used a high-fat diet (HFD)-induced NAFLD mouse model. Decreased levels of CerS2 and increased levels of CerS6 were observed in the steatotic livers of mice fed a HFD. In vitro experiments with Hep3B cells indicated the protective role of CerS2 and the detrimental role of CerS6 in the ER stress response induced by palmitate treatment. In particular, CerS6 overexpression increased sterol regulatory element-binding protein-1 (SREBP-1) cleavage with decreased levels of INSIG-1, leading to increased lipogenesis. Blocking ER stress abrogated the detrimental effects of CerS6 on palmitate-induced SREBP-1 cleavage. In accordance with the protective role of CerS2 in the palmitate-induced ER stress response, CerS2 knockdown enhanced ER stress and SREBP-1 cleavage, and CerS2 heterozygote livers exhibited a stronger ER stress response and higher triglyceride levels following HFD. Finally, treatment with a low dose of bortezomib increased hepatic CerS2 expression and protected the development of NAFLD following HFD. These results indicate that CerS and its derivatives impact hepatic ER stress and lipogenesis differently and might be therapeutic targets for NAFLD., Liver disease: Boosting a protective protein Promoting the activity of a protective membrane protein may help limit the development of non-alcoholic fatty liver disease (NAFLD) in obesity. Stress on a key cellular organelle, the endoplasmic reticulum (ER), contributes to NAFLD progression. Woo-Jae Park at Gachon University in Incheon, Joo-Won Park at Ewha Womans University, Seoul, and co-workers across South Korea have uncovered the role of a family of ER membrane proteins called ceramide synthases (CerS) in the regulation of ER stress during disease development. The team found increased levels of CerS6 in the livers of mouse fed a high-fat diet, while CerS2 decreased. The increased C16-ceramide by CerS6 overexpression triggered excess fat formation by increasing ER stress and SREBP-1 cleavage. However, when the team enhanced the expression of CerS2 using an existing chemotherapy drug, mice were protected from developing NAFLD.

The important membrane lipid, ceramide, is generated by a family of homologous enzymes, the ceramide synthases (CerSs), multi-spanning membrane proteins located in the endoplasmic reticulum. Six CerS isoforms exist in mammals with each using a subset of acyl-CoAs for (dihydro)ceramide synthesis. A number of mice have been generated in which one or other CerS has been genetically manipulated, including complete knock-outs, with each displaying phenotypes concomitant with the expression levels of the CerS in question and the presumed biological function of the ceramide species that it generates. We recently described a short C-terminal motif in the CerS which is involved in CerS dimer formation; deleting this motif had no effect on the ability of the CerS to synthesize ceramide in vitro. In the current study, we generated a CerS6 mouse using CRISPR-Cas9, in which the DDRSDIE motif was replaced by ADAAAIA. While levels of CerS6ADAAAIA expression were unaffected in the CerS6ADAAAIA mouse, and CerS6ADAAAIA was able to generate C16-ceramide in vitro, ceramide levels were significantly reduced in the CerS6ADAAAIA mouse, suggesting that replacing this motif affects an as-yet unknown mechanism of regulation of ceramide synthesis via the DDRSDIE motif in vivo. Crossing CerS6ADAAAIA mice with CerS5 null mice led to generation of viable mice in which C16-ceramide levels were reduced by up to 90%, suggesting that depletion of C16-ceramide levels is compensated for by other ceramide species with different acyl chain lengths.

(1) Background: six mammalian ceramide synthases (CerS1–6) determine the acyl chain length of sphingolipids (SLs). Although ceramide levels are increased in murine allergic asthma models and in asthmatic patients, the precise role of SLs with specific chain lengths is still unclear. The role of CerS2, which mainly synthesizes C22–C24 ceramides, was investigated in immune responses elicited by airway inflammation using CerS2 null mice. (2) Methods: asthma was induced in wild type (WT) and CerS2 null mice with ovalbumin (OVA), and inflammatory cytokines and CD4 (cluster of differentiation 4)+ T helper (Th) cell profiles were analyzed. We also compared the functional capacity of CD4+ T cells isolated from WT and CerS2 null mice. (3) Results: CerS2 null mice exhibited milder symptoms and lower Th2 responses than WT mice after OVA exposure. CerS2 null CD4+ T cells showed impaired Th2 and increased Th17 responses with concomitant higher T cell receptor (TCR) signal strength after TCR stimulation. Notably, increased Th17 responses of CerS2 null CD4+ T cells appeared only in TCR-mediated, but not in TCR-independent, treatment. (4) Conclusions: altered Th2/Th17 immune response with higher TCR signal strength was observed in CerS2 null CD4+ T cells upon TCR stimulation. CerS2 and very-long chain SLs may be therapeutic targets for Th2-related diseases such as asthma.

Gaucher disease (GD) is currently the focus of considerable attention due primarily to the association between the gene that causes GD (GBA) and Parkinson's disease. Mouse models exist for the systemic (type 1) and for the acute neuronopathic forms (type 2) of GD. Here we report the generation of a mouse that phenotypically models chronic neuronopathic type 3 GD. Gba-/-;Gbatg mice, which contain a Gba transgene regulated by doxycycline, accumulate moderate levels of the offending substrate in GD, glucosylceramide, and live for up to 10 months, i.e. significantly longer than mice which model type 2 GD. Gba-/-;Gbatg mice display behavioral abnormalities at ∼4 months, which deteriorate with age, along with significant neuropathology including loss of Purkinje neurons. Gene expression is altered in the brain and in isolated microglia, although the changes in gene expression are less extensive than in mice modeling type 2 disease. Finally, bone deformities are consistent with the Gba-/-;Gbatg mice being a genuine type 3 GD model. Together, the Gba-/-;Gbatg mice share pathological pathways with acute neuronopathic GD mice but also display differences that might help understand the distinct disease course and progression of type 2 and 3 patients.

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

Ceramide synthases (CerS) synthesise ceramides of defined acyl chain lengths, which are thought to mediate cellular processes in a chain length-dependent manner. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), we observed a significant elevation of CerS2 and its products, C24-ceramides, in CD11b(+) cells (monocytes and neutrophils) isolated from blood. This result correlates with the clinical finding that CerS2 mRNA expression and C24-ceramide levels were significantly increased by 2.2- and 1.5-fold, respectively, in white blood cells of MS patients. The increased CerS2 mRNA/C24-ceramide expression in neutrophils/monocytes seems to mediate pro-inflammatory effects, since a specific genetic deletion of CerS2 in blood cells or a total genetic deletion of CerS2 significantly delayed the onset of clinical symptoms, due to a reduced infiltration of immune cells, in particular neutrophils, into the central nervous system. CXCR2 chemokine receptors, expressed on neutrophils, promote the migration of neutrophils into the central nervous system, which is a prerequisite for the recruitment of further immune cells and the inflammatory process that leads to the development of MS. Interestingly, neutrophils isolated from CerS2 null EAE mice, as opposed to WT EAE mice, were characterised by significantly lower CXCR2 receptor mRNA expression resulting in their reduced migratory capacity towards CXCL2. Most importantly, G-CSF-induced CXCR2 expression was significantly reduced in CerS2 null neutrophils and their migratory capacity was significantly impaired. In conclusion, our data strongly indicate that G-CSF-induced CXCR2 expression is regulated in a CerS2-dependent manner and that CerS2 thereby promotes the migration of neutrophils, thus, contributing to inflammation and the development of EAE and MS.

Ceramide synthase 2 (CerS2) null mice cannot synthesize very-long acyl chain (C22-C24) ceramides resulting in significant alterations in the acyl chain composition of sphingolipids. We now demonstrate that hepatic triacylglycerol (TG) levels are reduced in the liver but not in the adipose tissue or skeletal muscle of the CerS2 null mouse, both before and after feeding with a high fat diet (HFD), where no weight gain was observed and large hepatic nodules appeared. Uptake of both BODIPY-palmitate and [VH]-palmitate was also abrogated in the hepa- tocytes and liver. The role of a number of key proteins involved in fatty acid uptake was examined, including FATP5, CD36/FAT, FABPpm and cytoplasmic FABP1. Levels of FATP5 and FABP1 were decreased in the CerS2 null mouse liver, whereas CD36/FAT levels were significantly elevated and CD36/FAT was also mislocalized upon insulin treatment. Moreover, treatment of hepatocytes with C22-C24-ceramides down-regulated CD36/FAT levels. Infection of CerS2 null mice with recombinant adeno-associated virus (rAAV)-CerS2 restored normal TG levels and corrected the mislocalization of CD36/FAT, but had no effect on the intracellular localization or levels of FATP5 or FABP1. Together, these results demonstrate that hepatic fatty acid uptake via CD36/FAT can be regulated by altering the acyl chain composition of sphingolipids.

Summary Inhibition of ceramide synthesis prevents diabetes, steatosis, and cardiovascular disease in rodents. Six different ceramide synthases (CerS) that differ in tissue distribution and substrate specificity account for the diversity in acyl-chain composition of distinct ceramide species. Haploinsufficiency for ceramide synthase 2 (CerS2), the dominant isoform in the liver that preferentially makes very-long-chain (C22/C24/C24:1) ceramides, led to compensatory increases in long-chain C16-ceramides and conferred susceptibility to diet-induced steatohepatitis and insulin resistance. Mechanistic studies revealed that these metabolic effects were likely due to impaired β-oxidation resulting from inactivation of electron transport chain components. Inhibiting global ceramide synthesis negated the effects of CerS2 haploinsufficiency in vivo, and increasing C16-ceramides by overexpressing CerS6 recapitulated the phenotype in isolated, primary hepatocytes. Collectively, these studies reveal that altering sphingolipid acylation patterns impacts hepatic steatosis and insulin sensitivity and identify CerS6 as a possible therapeutic target for treating metabolic diseases associated with obesity.

Sphingolipids are important structural components of cell membranes and act as critical regulators of cell function by modulating intracellular signaling pathways. Specific sphingolipids, such as ceramide, glucosylceramide, and ganglioside GM3, have been implicated in various aspects of insulin resistance, because they have been shown to modify several steps in the insulin signaling pathway, such as phosphorylation of either protein kinase B (Akt) or of the insulin receptor. We now explore the role of the ceramide acyl chain length in insulin signaling by using a ceramide synthase 2 (CerS2) null mouse, which is unable to synthesize very long acyl chain (C22-C24) ceramides. CerS2 null mice exhibited glucose intolerance despite normal insulin secretion from the pancreas. Both insulin receptor and Akt phosphorylation were abrogated in liver, but not in adipose tissue or in skeletal muscle. The lack of insulin receptor phosphorylation in liver correlated with its inability to translocate into detergent-resistant membranes (DRMs). Moreover, DRMs in CerS2 null mice displayed properties significantly different from those in wild-type mice, suggesting that the altered sphingolipid acyl chain length directly affects insulin receptor translocation and subsequent signaling. Conclusion: We conclude that the sphingolipid acyl chain composition of liver regulates insulin signaling by modifying insulin receptor translocation into membrane microdomains. (HEPATOLOGY 2013)

Little is known about the effects of altering sphingolipid (SL) acyl chain structure and composition on the biophysical properties of biological membranes. We explored the biophysical consequences of depleting very long acyl chain (VLC) SLs in membranes prepared from lipid fractions isolated from a ceramide synthase 2 (CerS2)-null mouse, which is unable to synthesize C22–C24 ceramides. We demonstrate that ablation of CerS2 has different effects on liver and brain, causing a significant alteration in the fluidity of the membrane and affecting the type and/or extent of the phases present in the membrane. These changes are a consequence of the depletion of VLC and unsaturated SLs, which occurs to a different extent in liver and brain. In addition, ablation of CerS2 causes changes in intrinsic membrane curvature, leading to strong morphological alterations that promote vesicle adhesion, membrane fusion, and tubule formation. Together, these results show that depletion of VLC-SLs strongly affects membrane biophysical properties, which may compromise cellular processes that critically depend on membrane structure, such as trafficking and sorting.

We have recently shown that phosphatidylcholine (PC) metabolism is altered in a macrophage model of Gaucher disease. We now demonstrate that treatment of macrophages with conduritol-B-epoxide (CBE), a glucocerebrosidase inhibitor, results in elevated activity of CTP:phosphocholine cytidylyltransferase (CCT), the rate-limiting enzyme in the pathway of PC biosynthesis. Furthermore, we provide evidence for a role for CCT in Gaucher macrophage growth by using macrophages derived from a genetically modified mouse which lacks a specific CCT isoform, CCTalpha, in macrophages. Upon CBE-treatment, macrophage size, analyzed by microscopy and by FACS, was significantly increased in macrophages from control mice, but did not increase, or increased to a much lower extent, in CCTalpha-/- macrophages. Together, these results suggest that the increase in PC biosynthesis is mediated via CCTalpha, and suggests a possible role for macrophage CCTalpha in Gaucher disease pathology.

Tumor necrosis factor receptor–associated factor (TRAF)2 is a critical adaptor molecule for tumor necrosis factor (TNF) receptors in inflammatory and immune signaling. Upon receptor engagement, TRAF2 is recruited to CD40 and translocates to lipid rafts in a RING finger-dependent process, which enables the activation of downstream signaling cascades including c-Jun NH2-terminal kinase (JNK) and nuclear factor (NF)-κB. Although TRAF1 can displace TRAF2 and CD40 from raft fractions, it promotes the ability of TRAF2 activate signaling over a sustained period of time. Removal of the RING finger of TRAF2 prevents its translocation into detergent-insoluble complexes and renders it dominant negative for signaling. TRAF1−/− dendritic cells show attenuated responses to secondary stimulation by TRAF2-dependent factors and increased stimulus-dependent TRAF2 degradation. Replacement of the RING finger of TRAF2 with a raft-targeting signal restores JNK activation and association with the cyto-skeletal protein Filamin, but not NF-κB activation. These findings offer insights into the mechanism of TRAF2 signaling and identify a physiological role for TRAF1 as a regulator of the subcellular localization of TRAF2.

Acid sphingomyelinase and ceramide have previously been shown to play a central role in infections with Neisseria gonorrhoeae, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli, and Mycobacterium avium. Recent studies have extended the role of sphingolipids in bacterial infections and have demonstrated that ceramide and sphingosine are central to the defense of lungs against bacterial pathogens. Ceramide accumulates in the airway epithelium of cystic fibrosis and ceramide synthase 2 (CerS2)-deficient mice, which respond to the lack of very long chain (C22-C24-) ceramides with a profound compensatory increase of long chain (mainly C16-) ceramides. In contrast, sphingosine is present in healthy airways and is almost completely absent from diseased or deficient epithelial cells. Both sphingolipids are crucially involved in the high susceptibility to infection of cystic fibrosis and CerS2-deficient mice, as indicated by findings showing that the normalization of ceramide and sphingosine levels rescue these mice from acute infection with P. aeruginosa.

Background & Aims Sortilin traffics newly synthesized molecules from the trans-Golgi apparatus along secretory pathways to endosomes, lysosomes or to the cell surface. Sortilin trafficking of acid sphingomyelinase (aSMase) may regulate ceramide levels, a major modulator of insulin signalling. We therefore tested whether sortilin deficiency reduces hepatic and adipose tissue aSMase activity, improving insulin sensitivity in diet-induced obesity (DIO). Methods DIO in C57BL/6 (WT) and sortilin −/− mice was induced by high-fat diet feeding for 10weeks. Results Sortilin −/− mice gained less body weight and less visceral fat, despite similar food intake compared to WT type mice and had enhanced glucose uptake in insulin tolerance tests, which was further corroborated by enhanced hepatic pAkt expression. Sortilin deficiency led to attenuated hepatic steatosis, reduced expression of genes involved in lipogenesis, ceramide synthesis and inflammatory cytokine production and reduced activity of ceramide synthase 5/6 (CerS5/6). Sortilin −/− mice had reduced hepatic aSMase activity under both steady-state and DIO. Likewise, sortilin −/− hepatocytes displayed hypersensitivity to insulin, due to enhanced insulin receptor downstream signalling. In adipose tissue, sortilin −/− mice exhibited lower expression of inflammatory cytokines and lower expression and activity of CerS5/6. As in liver, adipose tissue displayed increased insulin signalling, accompanied by attenuated aSMase activity. Conclusions Sortilin deficiency induces a beneficial metabolic phenotype in liver and adipose tissue upon DIO, mediated in part by reduced aSMase activity.

Mice whose IgH alleles are engineered to encode two distinct antibody heavy (H) chains generate a normal-sized B cell compartment in which most cells stably express the two heavy chains. This demonstrates that “toxicity” of bi-allelic H chain expression and cell- autonomous mechanisms of silencing in-frame IgH gene rearrangements do not significantly contribute to allelic exclusion at the IgH locus. Notwithstanding, the stability of the various engineered IgH loci during B cell development in the bone marrow differed substantially from each other.

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.

Rapid conduction of nerve impulses requires coating of axons by myelin sheaths, which are multilamellar, lipid-rich membranes produced by oligodendrocytes in the central nervous system. To act as an insulator, myelin has to form a stable and firm membrane structure. In this study, we have analyzed the biophysical properties of myelin membranes prepared from wild-type mice and from mouse mutants that are unable to form stable myelin. Using C-Laurdan and fluorescence correlation spectroscopy, we find that lipids are tightly organized and highly ordered in myelin isolated from wild-type mice, but not from shiverer and ceramide synthase 2 null mice. Furthermore, only myelin lipids from wild-type mice laterally segregate into physically distinct lipid phases in giant unilamellar vesicles in a process that requires very long chain glycosphingolipids. Taken together, our findings suggest that oligodendrocytes exploit the potential of lipids to self-segregate to generate a highly ordered membrane for electrical insulation of axons.

Sphingolipids (SLs) act as signaling molecules and as structural components in both neuronal cells and myelin. We now characterize the biochemical, histological, and behavioral abnormalities in the brain of a mouse lacking very long acyl (C22–C24) chain SLs. This mouse, which is defective in the ability to synthesize C22–C24-SLs due to ablation of ceramide synthase 2, has reduced levels of galactosylceramide (GalCer), a major component of myelin, and in particular reduced levels of non-hydroxy-C22–C24-GalCer and 2-hydroxy-C22–C24- GalCer. Noteworthy brain lesions develop with a time course consistent with a vital role for C22–C24-GalCer in myelin stability. Myelin degeneration and detachment was observed as was abnormal motor behavior originating from a subcortical region. Additional abnormalities included bilateral and symmetrical vacuolization and gliosis in specific brain areas, which corresponded to some extent to the pattern of ceramide synthase 2 expression, with astrogliosis considerably more pronounced than microglial activation. Unexpectedly, unidentified storage materials were detected in lysosomes of astrocytes, reminiscent of the accumulation that occurs in lysosomal storage disorders. Together, our data demonstrate a key role in the brain for SLs containing very long acyl chains and in particular GalCer with a reduction in their levels leading to distinctive morphological abnormalities in defined brain regions.

We have generated a mouse that cannot synthesize very long acyl chain (C22-C24) ceramides (Pewzner-Jung, Y., Park, H., Laviad, E. L., Silva, L. C., Lahiri, S., Stiban, J., Erez-Roman, R., Brugger, B., Sachsenheimer, T., Wieland, F. T., Prieto, M., Merrill, A. H., and Futerman, A. H. (2010) J. Biol. Chem. 285, 10902-10910) due to ablation of ceramide synthase 2 (CerS2). As a result, significant changes were observed in the sphingolipid profile of livers from these mice, including elevated C16-ceramide and sphinganine levels. We now examine the functional consequences of these changes. CerS2 null mice develop severe nonzonal hepatopathy from about 30 days of age, the age at which CerS2 expression peaks in wild type mice, and display increased rates of hepatocyte apoptosis and proliferation. In older mice there is extensive and pronounced hepatocellular anisocytosis with widespread formation of nodules of regenerative hepatocellular hyperplasia. Progressive hepatomegaly and noninvasive hepatocellular carcinoma are also seen from approximately 10 months of age. Even though CerS2 is found at equally high mRNA levels in kidney and liver, there are no changes in renal function and no pathological changes in the kidney. High throughput analysis of RNA expression in liver revealed up-regulation of genes associated with cell cycle regulation, protein transport, cell-cell interactions and apoptosis, and down-regulation of genes associated with intermediary metabolism, such as lipid and steroid metabolism, adipocyte signaling, and amino acid metabolism. In addition, levels of the cell cycle regulator, the cyclin dependent-kinase inhibitor p21(WAF1/CIP1), were highly elevated, which occurs by at least two mechanisms, one of which may involve p53. We propose a functional rationale for the synthesis of sphingolipids with very long acyl chains in liver homeostasis and in cell physiology.

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.

Beyond its established function in hematopoiesis, the bone marrow hosts mature lymphocytes and acts as a secondary lymphoid organ in the initiation of T cell and B cell responses. Here we report the characterization of bone marrow-resident dendritic cells (bmDCs). Multiphoton imaging showed that bmDCs were organized into perivascular clusters that enveloped blood vessels and were seeded with mature B lymphocytes and T lymphocytes. Conditional ablation of bmDCs in these bone marrow immune niches led to the specific loss of mature B cells, a phenotype that could be reversed by overexpression of the antiapoptotic factor Bcl-2 in B cells. The presence of bmDCs promoted the survival of recirculating B cells in the bone marrow through the production of macrophage migration-inhibitory factor. Thus, bmDCs are critical for the maintenance of recirculating B cells in the bone marrow.

Knockout of caspase-8, a cysteine protease that participates in the signaling for cell death by receptors of the TNF/nerve growth factor family, is lethal to mice in utero. To explore tissue-specific roles of this enzyme, we established its conditional knockout using the Cre/loxP recombination system. Consistent with its role in cell death induction, deletion of caspase-8 in hepatocytes protected them from Fas-induced caspase activation and death. However, application of the conditional knockout approach to investigate the cause of death of caspase-8 knockout embryos revealed that this enzyme also serves cellular functions that are nonapoptotic. Its deletion in endothelial cells resulted in degeneration of the yolk sac vasculature and embryonal death due to circulatory failure. Caspase-8 deletion in bone-marrow cells resulted in arrest of hemopoietic progenitor functioning, and in cells of the myelomonocytic lineage, its deletion led to arrest of differentiation into macrophages and to cell death. Thus, besides participating in cell death induction by receptors of the TNF/nerve growth factor family, caspase-8, apparently independently of these receptors, also mediates nonapoptotic and perhaps even antiapoptotic activities.

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

Increases in ceramide levels have been implicated in the pathogenesis of both acute or chronic lung injury models. However, the role of individual ceramide species, or of the enzymes that are responsible for their synthesis, in lung health and disease has not been clarified. We now show that C24- and C16-ceramides are the most abundant lung ceramide species, paralleled by high expression of their synthetic enzymes, ceramide synthase 2 (CerS2) and CerS5, respectively. Furthermore, the ceramide species synthesis in the lung is homeostatically regulated, since mice lacking very long acyl chain C24-ceramides due to genetic deficiency of CerS2 displayed a ten-fold increase in C16-ceramides and C16-dihydroceramides along with elevation of acid sphingomyelinase and CerS5 activities. Despite relatively preserved total lung ceramide levels, inhibition of de novo sphingolipid synthesis at the level of CerS2 was associated with significant airflow obstruction, airway inflammation, and increased lung volumes. Our results suggest that ceramide species homeostasis is crucial for lung health and that CerS2 dysfunction may predispose to inflammatory airway and airspace diseases.