Ceramide synthases as potential targets for therapeutic intervention in human diseases

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Biochimica et Biophysica Acta xxx (2013) xxx–xxx

BBAMCB-57506; No. of pages: 11; 4C: 3, 4

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta

j ourna l homepage: www.e lsev ie r .com/ locate /bba l ip

Review

Ceramide synthases as potential targets for therapeutic intervention inhuman diseases☆

OFJoo-Won Park a, Woo-Jae Park b,c, Anthony H. Futerman b,⁎

a Department of Biochemistry, School of Medicine, Ewha Womans University, Seoul 158-710, South Koreab Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israelc Department of Biochemistry, School of Medicine, Gachon University, Incheon 406-799, South Korea

Abbreviations: CerS, ceramide synthase; DRMs, deterglong chain base; GalCer, galactosylceramide; HexCer, hexTNF, tumor necrosis factor☆ This article is part of a Special Issue entitled New fron⁎ Corresponding author. Tel.: +972 8 9342704; fax: +

E-mail address: [email protected] (A.H. F

1388-1981/$ – see front matter © 2013 Published by Elsehttp://dx.doi.org/10.1016/j.bbalip.2013.08.019

Please cite this article as: J.-W. Park, et al.,Biophys. Acta (2013), http://dx.doi.org/10.10

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Article history:Received 30 July 2013Received in revised form 28 August 2013Accepted 29 August 2013Available online xxxx

Keywords:SphingolipidCeramide synthaseAcyl chain lengthDiseaseSpecificityTherapeutic target

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RCeramide is located at a key hub in the sphingolipidmetabolic pathway and also acts as an important cellular sig-nalingmolecule. Ceramide contains one acyl chain which is attached to a sphingoid long chain base via an amidebond, with the acyl chain varying in length and degree of saturation. The identification of a family of sixmamma-lian ceramide synthases (CerS) that synthesize ceramidewith distinct acyl chains, has led to significant advancesin our understanding of ceramide biology, including further delineation of the role of ceramide in various path-ophysiologies in both mice and humans. Since ceramides, and the complex sphingolipids generated from cer-amide, are implicated in disease, the CerS might potentially be novel targets for therapeutic intervention in thediseases inwhich the ceramide acyl chain length is altered. This article is part of a Special Issue entitled New fron-tiers in sphingolipid biology.

© 2013 Published by Elsevier B.V.

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1. Introduction

Upon completion of the human genome project in 2003, significantadvances in medicine were promised. Whether these promises havebeen fulfilled is a matter of ongoing debate, but what has becomeclear is that the post-genomic era is characterized by more attentionbeing paid to metabolomics, proteomics, glycomics and lipidomics. Interms of lipidomics, advances in mass spectrometry techniques haveled to a revolution in our understanding of the complexity of lipids,both in terms of their structural complexity and their distinct functions[1,2]. However, while lipidomics has generated, justifiably, great atten-tion, significant gaps still remain in our knowledge of relatively simplebiochemical processes. Among these are the study of the biochemistryand regulation of sphingolipid (SL) synthesis and degradation. Surpris-ingly, themolecular identification of the enzymes involved in SLmetab-olismwas only completed in the past few years [3]. Alongwith this, hascome the realization that lipids, and in particular SLs, are involved in anumber of human diseases, either as the direct cause (such as in thesphingolipidoses [4,5]) or as up- or down-stream components [6].Irrespective of the precise roles that SLs play in human disease, it has

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ent-resistant membranes; LCB,osylceramide; SL, sphingolipid;

tiers in sphingolipid biology.972 8 9344112.uterman).

vier B.V.

Ceramide synthases as poten16/j.bbalip.2013.08.019

become apparent that intervention in the SL metabolic pathwayshows great promise as the target of novel therapeutic paradigms.

In the current review, wewill focus on ceramide, the chemical back-bone of all SLs, and an important cellular messenger, and in particularon the ceramide synthases (CerS), the recently-identified enzymesthat are responsible for ceramide synthesis. Our focus will be on the in-volvement of ceramides with distinct acyl chains in human disease, andthe possible therapeutic potential of ceramide and CerS.

2. Biochemical characterization of CerS

2.1. An overview of SL synthesis

SLs consist of a sphingoid long chain base to which a fatty acid isN-acylated to forman amide bond at the C2 position, and various polar ornon-polar head groups at theC1position. SL synthesis begins in the endo-plasmic reticulum with the condensation of L-serine and palmitoyl CoA,generating 3-ketosphinganine [7], although other amino acids, such asglycine and alanine, and other fatty acids, such as myristic acid, can actas substrates for serine palmitoyl transferase [8,9]. 3-ketosphinganineis then reduced to sphinganine. N-acylation of sphinganine by CerSgenerates dihydroceramide, which is subsequently reduced to ceramide.Ceramide is the backbone of all SLs and is metabolized to complexSLs such as sphingomyelin (SM) (by addition of phosphorylcholine),glucosylceramide (GlcCer) (by addition of glucose), galactosylceramide(GalCer) (by addition of galactose), ceramide-1-phosphate (by phos-phorylation) and sphingosine 1-phosphate (S1P) (via degradation of

tial targets for therapeutic intervention in human diseases, Biochim.

http://dx.doi.org/10.1016/j.bbalip.2013.08.019

mailto:[email protected]


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Table 2 t2:1

t2:2Antibodies used for study of CerS tissue distribution.

t2:3CerS Commercial Non-commercial References

t2:4company(catalog number)

Detectedspecies

Source of antibody Detectedspecies

t2:5CerS1 Ginkel et al.a Mouse [51]t2:6CerS2 Sigma-Aldrich

Corporation Q2Mouse,human

Kremser et al.c Mouse [27]

t2:7(HPA027262)b

t2:8CerS3 Acris Rat, mouse Jennemann et al.e Mouse [66,28]t2:9(AP16822PU-N)d

t2:10CerS6 Ebel et al.f Mouse [29]

a Against the C terminus of mouse CerS1 (QMRELEDLREYDTLEAQ). t2:11b Against the human peptide TPLAALLNIKEKTRLRAPPNATLEHFYLTSGKQPKQVEVELL

SRQSGLSGRQVARWFRRRRNQDRPSLLKKFREA. t2:12c Against a C-terminal peptide (SRLLANGHPILNNNHPKND). t2:13d Against a C-terminal peptide (RAERHLIPNGQHGH) of human CerS3. t2:14e Against a peptide located at the C-terminus of mouse CerS3. t2:15f Against a C-terminal peptide CDDEDSEPPGKKPH. t2:16

2 J.-W. Park et al. / Biochimica et Biophysica Acta xxx (2013) xxx–xxx

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ceramide to sphingosine and subsequent phosphorylation). Complex SLscan also be hydrolyzed and recycled by the salvage pathway [10], andsphingosine, which is generated in this pathway, is also a substrate forthe CerS.

2.2. Substrate specificity and structural features of the CerS

SixmammalianCerS exist (CerS1-6),with each encodedonadifferentchromosome [7,11]. Each CerS displays specificity toward fatty acyl CoAsof defined chain length (Table 1), although the hydroxylation and satura-tion status of the acyl CoAs does not appear to influence their specificity[12,13]. The Km values for sphinganine range from ~2 μM (CerS3) to5 μM (CerS2) [14], but the CerS are less specific towards the LCB than to-wards acyl CoAs, since they are able toN-acylate a variety of LCBs, such asnatural LCBs, i.e., sphinganine, sphingosine and phytosphingosine [15],LCB analogs, i.e., fumonisin B1 and hydrolyzed fumonisins [16,17], and afluorescent analog of sphinganine, NBD-sphinganine [18]. Recently, ad16:0-sphingoid base, which is formed by utilization of myristoyl-CoAby serine palmitoyl transferase, was also shown to be a CerS substrate,with CerS1 preferentially utilizing a d16:0-sphingoid base compared tothe canonical d18:0-sphinoid base [19].

In terms of their structural features, all six CerS contain a domaincalled the Tram-Lag-CLN8 (TLC) domain. A minimum region in theTLC domain required for CerS acyl CoA specificity was recently identi-fied [7,20], although the exact residues that determine specificity havenot been delineated. For CerS activity, the Lag1p motif, a conservedstretch of 52 amino acids within the TLC domain, is essential [21,22]and the terminal 12 amino acid residues of the Hox-like domain playan important role in CerS5 and CerS6 activity [23]. The three-dimensional structure of the CerS, and their precise membrane topolo-gy, remain to be elucidated.

2.3. Tissue and cellular distribution of CerS

Mammalian CerS exhibit a distinct tissue distribution pattern [24,25](Table 1). Briefly, CerS1 is mainly expressed in brain and skeletal mus-cle, CerS2 is highly expressed in lung, liver and kidney, CerS3 isexpressed in testis and skin and CerS4 is widely expressed but mainlyfound in heart and in leukocytes [13,24,25]. CerS5 and 6 are expressedin relatively low amounts, with CerS5 expressed in prostate and skeletalmuscle and CerS6 expressed in intestine and lymph node [13]. Recently,reliable antibodies for a number of CerS have become available(Table 2), allowing precise localization of individual CerS [26,27]. For in-stance, CerS3 is expressed in the upper stratum spinosum and stratumgranulosumof the skin [28], andCerS6 is expressed in the hippocampus,Purkinje cell layer of the cerebellum, and glomeruli in the kidney [29].

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Table 1CerS and related human pathologies and diseases.

CerS Fatty acid specificity of CerS Tissue distributiona HC

CerS1 C18 Brain (neurons), cerebellum H(Purkinje cells), skeletal muscle

CerS2 C22–C24 Brain (oligodendrocytes) lung, liver, intestineadrenal gland, kidney white adipose tissues

B

CerS3 C26 Skin, testis CCerS4 C18–C22 Heart, skin BCerS5 C16 C

CerS6 C14, C16 Brain (hippocampus), kidney (glomeruli),small and large intestine, thymus

BCM

a Tissue distribution is mainly taken from studies in mice [27,29,30,51,66,121].b References refer to the human disease in which these changes are observed.

Please cite this article as: J.-W. Park, et al., Ceramide synthases as potenBiophys. Acta (2013), http://dx.doi.org/10.1016/j.bbalip.2013.08.019

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CerS2, the most abundant CerS, at least with respect to its mRNA levels[24], is expressed in a cell type-specific manner; for example, in liver,CerS2 is expressed in hepatocytes but not in Ito or in Kupffer cells[27]. In brain, CerS2 is expressed in oligodendrocytes but not in neurons[27]. However, ceramide levels with distinct acyl chain lengths do notalways correlate with CerS expression [24,26,30]; thus, despite highCerS2 expression in small intestine, C16- and C18-ceramides are themajor ceramide species [26]. Therefore, other factors apart from CerSexpression and activity levels determine the acyl chain length composi-tion of ceramides in a particular tissue.

2.4. Regulation of CerS

Little is currently known about CerS regulation in vivo [31], but anumber of observations suggestmultiple levels of regulation. For exam-ple, selective knockdown of CerS6 in MCF-7 human breast adenocarci-noma cells increased CerS5 expression [31], and C16-ceramide levelswere elevated in the liver of CerS2 null mouse, although there was nosignificant elevation in CerS5 or 6 activity [32]. CerS enzyme activitydoes not always correlate with CerS mRNA and protein expres-sion [27,30], possibly due to post-translational modifications. CerS2, 5and 6 can be N-glycosylated at the N-terminal region [33], and CerS1can be phosphorylated by protein kinase C at putative serine/threoninephosphorylation sites, which affects the translocation of CerS1 from theER to the Golgi apparatus [34]. Although glycosylation does not affectCerS enzymeactivity [33], phosphorylationmay play a role in regulatingCerS activity [35]. Thus, the AGC kinase, YpK2, a downstream kinase of

uman disease associated with change inerS or ceramide acyl chain length

Changes in SL or CerS levels Referencesb

ead and neck squamous carcinoma C16-Cer↑ [81,83]C18-Cer↓

reast cancer CerS2↑ [84,86]C24-Cer↑C24:1-Cer↑

ongenital ichthyosis C26–C34-Cer↓ [65,66]reast cancer CerS4↑ [86]ardiomyohypertrophy C14-Cer↑ [92]

C18:1-Cer↑reast cancer CerS6↑ [84,86]olon cancer C16-Cer↑ultiple sclerosis CerS6↓

C16-Cer↓ [88,89]CerS6↑C16-Cer↑ [94]



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TORC2 (target of rapamycin complex 2), activates de novo ceramidesynthesis in Saccharomyces cerevisiae, which is antagonized by theCa2+/calmodulin-dependent phosphatase, calcineurin [36]. p38 MAPkinase increases CerS1 ubiquitination whereas protein kinase C de-creases the proteasomal turnover of CerS1. The C-Jun N-terminal kinase3 signaling pathway plays a role in ischemia–reperfusion induced cer-amide generation by activating CerS [37]. In addition, CerS1, CerS2 andCerS5 were predicted to be phosphorylated by a large-scale phosphory-lation analysis [38].

CerS activity can also be regulated by direct molecular binding. Co-immunoprecipitation experiments demonstrated the existence of CerShetero-complexes [39] and CerS dimer formation modulates CerS en-zyme activity [40]. CerS2 activity is enhanced by co-expression withcatalytically-active forms of CerS5 or CerS6. Moreover, upon generationof a constitutive dimer, it was shown that the activity of CerS2 dependson the catalytic activity of CerS5 [40]. The activity of CerS6 was not af-fected by other CerS [41]. Thus, the combinatorial pattern of expression,and mode of interaction of CerS in any particular tissue might definewhich acyl chain species are formed in that tissue.

In yeast, the CerS homologs, Lag1p and Lac1p, require another pro-tein, Lip1 for their activity [42]. In contrast, there is currently no evidencethat mammalian CerS require another subunit [43]. However, other pro-teins do appear to interact, either directly or indirectly, with the CerS.Thus, elongation of very-long chain fatty acid protein (ELOVL1) is re-quired for full CerS2 activity [44], and Bak was postulated to form a stoi-chiometric complex with CerS4, 5 and 6 [45]. Lipids can also modulateCerS activity. Sphingosine-1-phosphate inhibits CerS2 activity by directinteraction via two residues that are part of a sphingosine-1-phosphatereceptor-like motif found only in CerS2 [24], and reconstituted CerS5 re-quires phosphatidylcholine for its activity [43].

3. Roles of SLs with defined fatty acid chain lengths

3.1. Specific roles of ceramides with defined acyl chain lengths in cellphysiology

Due to the discovery of the CerS, and due to advances in lipidomics, ithas become apparent that ceramides and SLs with different acyl chain

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Fig. 1. The functional roles of CerS2 in liver. Ablation of CerS2 depletes very-long acyl chain SLsacyl chain SLs changesmembrane properties, which disruptmembrane domains and cause altertranslocation into detergent resistantmembranes), fatty acid uptake (byCD36mislocalization aIncreasedC16-ceramide and sphinganine levels inhibitmitochondrial respiratory complex IV,wsynthase; Cx32, connexin 32; FATP5, fatty acid transport protein 5; IR, insulin receptor; ROS, re


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lengths play widely different roles in physiology and pathophysiology.In relation to human disease, one of the first examples that combinedstudy of the CerS and of the ceramides that were generated by specificCerS, was the observation that C18- and C16-ceramides, generated byCerS1 and CerS5 respectively, play opposing roles as pro-apoptoticand pro-survival molecules in head and neck squamous cell carcinomas[46]. At the cellular level, C16-ceramide [46] and C24:0/C24:1-ceramide[39,47] are considered pro- and anti-apoptotic, respectively, and a shiftin SL composition from C24 to C16 increases susceptibility to apoptosis[48]. At the molecular level, C16-ceramide but not C24-ceramide, in-hibits mitochondrial complex IV activity which causes oxidative stress[49] (Fig. 1).

Over the past 3–4 years, more evidence for specific roles ofceramides with defined acyl chain lengths has come from the study ofCerS null mice, and from the findings that the loss of a specific CerS can-not be compensated for by the activity of another CerS, leading to dis-tinct pathophysiologies in all of the CerS null mice that have beengenerated to date. Interestingly, in some cases, total ceramide levelsare unaltered in various tissues, by an as yet unknownmolecularmech-anism, presumably to maintain total cellular amounts of ceramide atan optimum level. For example, in CerS2 null mouse liver, total cer-amide levels are unaltered since C16-ceramide levels are increasedupon loss of C22–C24-ceramides [32]; however, despite attempts tocompensate for the loss of C22–C24-ceramides, themice develop severehepatopathy. Likewise, a CerS1 null mouse also displays elevated levelsof C16- and C22-ceramides upon depletion of C18-ceramide [50,51].Thus, although cells attempt to overcome the loss of specific ceramidespecies, they fall short inasmuch as depletion of any one specific cer-amide species leads to severe pathology.

3.2. Biophysical changes upon altering the SL acyl chain length

A significant body of evidence exists using model membranes, inwhich the effects of different ceramide species onmembrane propertieshave been examined [52,53]. For instance,modelmembranes consistingof phosphatidylcholine and ceramides with different acyl chains, dis-play widely different properties inasmuch as saturated ceramideshave a stronger impact on the fluid membrane, increase its order, and

(C22–C24), and C16-SLs and sphinganine levels are increased [32]. Depletion of very-longedmembrane properties, which affects insulin resistance (by inhibition of insulin receptornd FATP5down-regulation) and gap junction dysfunction (byCx32mislocalization) [58].1,2

hich causes chronic oxidative stress [49]. CD36; cluster of differentiation 36; CerS, ceramideactive oxygen species; Sa, sphinganine; SL, sphingolipid.



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Fig. 2. Insulin receptor translocation in CerS2 null mouse liver. Membrane domains in WT liver are tightly packed, permitting translocation of the insulin receptor. However, membranedomains in CerS2 null mouse liver are disrupted and insulin receptor translocation and phosphorylation are abrogated [58]. IR, insulin receptor; SL, sphingolipid.


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promote gel/fluid phase separation, while their unsaturated counter-parts have a lower or no ability to form gel domains [54]. Very-longchain ceramides form tubular structures likely due to their ability toform interdigitated phases [54]. When the effects of ceramides withvarying saturated acyl chains (C16–C24) were studied on the formationand stability of ordered domains, low amounts of C16-ceramide had astronger impact on the melting profile of the mixtures compared toC18-ceramide, suggesting that C16-ceramide gel domains were biggerand more stable than those formed by C18-ceramide, which can beexplained by the increase in ceramide acyl chain length giving rise toan increase in the average area per molecule [54,55].

In addition to studies on model membranes, altered membrane bio-physical properties have also been found in biological membranes isolat-ed fromCerS2 nullmice. Thus, CerS2 nullmouse livermembranes displayhigher membrane fluidity and morphological changes [32], including al-terations of intrinsicmembrane curvature, which promotes vesicle adhe-sion, membrane fusion, and tubule formation, along with alterations inthe type and/or extent of the phase present in the membrane [56,57].In addition to direct changes in membrane structure, measured by appli-cation of fluorescent probes on isolated membrane lipids, alteringthe acyl chain composition also significantly affects the properties ofdetergent-resistant membranes (DRMs) [58]. Although DRMs are some-what controversial, they do nevertheless provide a simple and quickmethod for determining some basic paradigms of membrane properties.DRMs isolated from CerS2 null mouse are disrupted, perhaps due to lossof interdigitation (Fig. 1). As a result, signaling pathways which dependuponmembrane properties, are disturbed. The best-characterized exam-ple is the case of the insulin receptor,which is not translocated intoDRMsupon insulin treatment (Fig. 2), resulting in a complete loss of insulin sig-naling [58]; similar results have been obtainedwith fatty acid uptake andgap junction mislocalization1,2 (Fig. 1).

1 WJ Park, JW Park, JR. Bame, AHMerrill Jr., J Storch, Y Pewzner-Jung and AH Futerman.A role for the sphingolipid acyl chain length in regulating triacylglycerol accumulation viamodulation of fatty acid uptake. Submitted for publication.

2 WJ Park, JWPark, R Erez-Roman, A Kogot-Levin, JR. Bame, B Tirosh, A Saada, AHMerrillJr., Y Pewzner-Jung and AH Futerman. Protection of a Ceramide synthase 2 null mousefrom drug-induced liver injury due to gap junction dysfunction caused by connexin 32mislocalization. Submitted for publication.


ED3.3. SL acyl chain length and endocytic trafficking disorders

Sincemembrane properties are altered, it might be expected that cel-lular functions that depend on membrane properties, such as cellulartrafficking, might be affected, and this is indeed the case. For instance,schlank, a Drosophila CerS homolog, regulates body fat storage [59] andits down-regulation led to defects in the endosomal trafficking of pro-teins [60]. CerS2 null mice display a similar phenotype in hepatocytes,since they display impaired fatty acid uptake due to mis-localization ofa fatty acid transport protein, CD36 (cluster of differentiation 36) protein,leading to reduced triglyceride levels in liver,1 and impaired TNF-α up-take due to defective TNF-α receptor internalization.3 The precisemolec-ular mechanism behind defective endocytosis upon depletion of very-long chain SLs is not known, but may be related to the disruption ofmembrane microdomains [58] in the plasma membrane and in earlyendosomes [61]. Similarly, in Arabidopsis, very-long acyl chain SLs arealso involved in trafficking pathways within specific endomembranecompartments [62].

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4. Mouse models of CerS deficiency

Over the past 3–4 years, a number of mice deficient in one or otherCerS have been generated. The first to be produced, and hence thebest-characterized, was a CerS2 null mouse [32,63,64]. Each CerS nullmouse displays an extremely different phenotype (Table 3), attestingto the importance and lack of redundancy of the CerS and of the cer-amide species that they generate. Moreover, in the one CerS mousemodel for which an equivalent human disease has been described(CerS3) [28,65,66], there is a significant overlap of phenotypes in themouse and in the human patients, suggesting that full characterizationof the mouse models will provide information germane to understand-ing the human diseases in which CerS deficiency is involved.

3 M Ali, J Fritsch, H Zigdon, Y Pewzner-Jung, S Schütze and AH Futerman. Altering acylchain composition of sphingolipids prevents LPS/GLN-mediated hepatic failure in miceby disrupting TNFR1 internalization. Submitted for publication.



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Table 3t3:1

t3:2 Characteristics of CerS null mouse models.

t3:3 Mouse Tissue Decreased SLs Increased SLs Phenotype References

t3:4 CerS1 null Brain C18-Cer C16-Cer Purkinje cell death in cerebellum [50,51]t3:5 HexCer C22-Cer Lipofuscin accumulationt3:6 Gangliosides Sphingosine Shrinkage and neuronal apoptosis in cerebellum and forebraint3:7 Sphinganine sphingosine1-P Cerebellar ataxiat3:8 Sphinganine1-P Myelin-associated glycoprotein ↓t3:9 CerS2 null Brain HexCer C18-HexCer Myelin sheath defects [64,67]t3:10 GalCer Sphinganine Vacuolization, gliosist3:11 Unidentified storage materials in lysosomes of astrocytest3:12 Lung C22–C24-Cer C16-Cer Airflow resistance ↑ [69]t3:13 DihydroCer Lung volume ↑t3:14 Sphinganine Foamy macrophage infiltrationt3:15 Inflammationt3:16 Liver C22–C24-Cer C16-SLs Hepatoadenoma [32,49,56,58,63,64]2

t3:17 Sphingosine Membrane fluidity ↑t3:18 Sphinganine High hepatocyte turnovert3:19 Altered cytoskeleton proteinst3:20 DRM disruptiont3:21 Hypoglycemia, insulin resistancet3:22 Mitochondrial dysfunctiont3:23 Altered gap junctionst3:24 Kidney C22–C24-Cer C16-Cer, SM No pathology [63,64]t3:25 C16-HexCert3:26 Sphingosinet3:27 Sphinganinet3:28 Adrenal gland C16-Cer, SM Lipofuscin 4

t3:29 Cer, SM Pheochromocytomat3:30 Sphingosinet3:31 Sphinganinet3:32 CerS3 null Skin ≥C26-Cer C16-Cer Lack of continuous extracellular lipid [28]t3:33 Cer GlcCer Lamellaet3:34 Death after birth due to transepidermal water losst3:35 Hyperkeratosis, deficient cornificationt3:36 Increased susceptibility to Candida albicans infectiont3:37 CerS6 null Brain Behavioral abnormalities including a clasping abnormality of their

hind limbs and a habituation deficit[29]

t3:38 Kidney C16-Cer C18-HexCer No pathologyt3:39 C16-SMt3:40 Thymus C16-Cer No pathologyt3:41 C16-HexCert3:42 Small intestine C16-Cer C20-HexCer No pathologyt3:43 Cert3:44 C16-SMt3:45 Large intestine C20-HexCer No pathology


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4.1. CerS1

CerS1 is mainly expressed in brain and skeletal muscle [13], andmost work on CerS1 has focused on the brain. Two CerS1 null micehave been generated [50,51], with little difference between them.CerS1 null mice show Purkinje cell death in the cerebellum, accumula-tion of lipofuscin and ubiquitylated proteins, shrinkage and neuronalapoptosis in the cerebellum and forebrain, and cerebellar ataxia dueto Purkinje cell death. As expected C18:0-ceramide levels were de-creased, with elevated C16- and C22-ceramide detected in one of themice [51], and sphingosine, sphinganine, sphingosine-1-phosphateand sphinganine-1-phosphate elevated in the brain [50,51]. Surprising-ly, gangliosides GM1, GD1a, GT1b and GD1b, along with myelin-associated glycoprotein, were decreased by ~60% in the cerebellumand forebrain [51]. The main conclusion of these studies is that C18-ceramide synthesis via CerS1 is of importance for Purkinje cell develop-ment in the forebrain and cerebellum.

4.2. CerS2

CerS2 null mice have been relatively well-characterized [32,49,58,63,67]. The major phenotypes described to date concern the brainand the liver, with myelin sheath defects in the former and chronichepatoadenoma, which progresses to hepatocellular carcinoma inolder animals, in the latter [32,63,64,67]. In both tissues, levels of long


chain ceramides are elevated (C18:0 and C18:1 in the brain and C16 inthe liver) to compensate for the loss of very-long chain ceramides. How-ever, in the brain, there is a significant reduction in GalCer levels [64,67],since very-long acyl chains are major components of brain GalCer;this loss of GalCer is the cause of the demyelination defects [67]. Addi-tional pathological features in the brain are vacuolization and gliosis inspecific brain areas, and accumulation of unidentified storage materialin lysosomes of astrocytes. Based on this data, CerS2 has been demon-strated to play an important role in GalCer formation andmyelin sheathmaintenance.

In the liver, high throughput analysis of RNA expression revealedup-regulation of genes associated with cell cycle regulation, protein trans-port, cell–cell interactions and apoptosis, and down-regulation ofgenes associated with intermediary metabolism, such as lipid and ste-roid metabolism, adipocyte signaling, and amino acid metabolism[63]. However, despite major changes in liver function and pathology,specific rather than general pathways of liver function are disrupted inCerS2 null mice, implying that altering the SL acyl chain composition af-fects specific biochemical events and cellular pathways, rather thancausing general liver dysfunction. For instance, CerS2 null mice exhibitglucose intolerance despite normal insulin secretion from the pancreas[58]. Both insulin receptor and Akt phosphorylation are abrogated, andthe lack of insulin receptor phosphorylation correlates with its inabilityto translocate into DRMs (Fig. 2). Likewise, CerS2 null mice are resistantto LPS/galactosamine-mediated fulminant hepatic failure, and cultured



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hepatocytes from CerS2 null mice were also insensitive to TNFα-mediated apoptosis due to the inability of CerS2 null mice to internalizethe TNF receptor,3 which is almost certainly related to the biophysicalchanges discussed above [32,56]. Finally, generation of reactive oxygenspecies is increased in liver due to hepatic mitochondrial complex IVdysfunction [49] and more specifically, due to C16-ceramide andsphinganine (which also accumulates in the CerS2 null mouse liver) di-rectly inhibiting complex IV activity of the mitochondria. [49] (Fig. 1).This latter effect is likely to be involved in the enhanced rates of apopto-sis seen in the CerS2 null mouse.

Both in the liver [63], and also in the adrenal gland,4 development ofcancerous tumors are observed. Cystic degeneration/spongiosis hepati-tis was observed in 20% of CerS2 null mice N8 months of age, and hepa-tocellular carcinoma was diagnosed in 30% of older mice [63]. Multipleceroids were detected in the x-zone (between the cortex and medullaof the adrenal gland), leading to pheochromocytoma in the majorityof 13–15 month-old CerS2 null mice.4 The pheochromocytoma mightbe caused by chronic oxidative stress as lipoxidation end-products arefound at high levels in ceroid [68].

Finally, CerS2 is also expressed at high levels in the lung, particularlyin the epithelial layers of the bronchi, rather than in the vascular endo-theliumor alveoli [69]. CerS2 nullmice lung display a 10-fold increase inC16-ceramide in the lung. These changes lead to airflow obstruction,airway inflammation and increased lung volumes [69].

4.3. CerS3

CerS3 is related to the development of germ cells [70] and isexpressed mainly in the testis and in the skin [24,71]. During the devel-opment of germ cells, levels of C26–C32–SLs increase concomitantlywith an increase in CerS3 expression [70]. A CerS3 null mouse displaysa complete loss of C26- and ultra-long chain (i.e., NC32) ceramides[28]. The mice show lack of continuous extracellular lipid lamellaeand a non-functional cornified lipid envelope, and as a result oftransepidermal water loss, CerS3 null mice die shortly after birth. Themice are also prone to Candida albicans infection because of the skin bar-rier disruption. In addition, CerS3 null mice display skin hyperkeratosisand deficient cornification.

4.4. CerS6

The most recent CerS null mouse to be described was a CerS6 nullmouse, and the phenotype of the mouse is the least dramatic of all theCerS null mice described to date [29]. As expected from the specificityand expression pattern of CerS6, C16–SLs were decreased in the kidneyand in the small intestine. Some behavioral abnormalities, including aclasping-abnormality of their hind limbs and a habituation deficit,were described, alongwith an increased density of microglia in the hip-pocampus. The relatively mild phenotype is probably related to the rel-atively low expression levels of CerS6 in most tissues [24], and possiblydue to the overlap of acyl CoA specificity with CerS5.

5. CerS in human diseases

Ceramides with defined acyl chain lengths are involved in a numberof humandiseases, and although there is less information about the spe-cific roles of CerS in human disease, it is very likely that CerS will also beimplicated in a number of diseases, directly via mutations in their cod-ing sequences (as has been reported for CerS3, see below) or indirectlyas a down-stream response to other metabolic alterations. Some select-ed examples of the roles of ceramide and CerS in human disease aregiven below.

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4 WJ Park, V Sasson, A Kogot-Levin, O Brenner, A Saada, H Park, AH Merrill Jr., YPewzner-Jung and AH Futerman. Spontaneous development of pheochromocytoma inCerS2 null mouse adrenal gland. Submitted for publication.


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5.1. Human patients with mutations in CerS genes

5.1.1. CerS2 single-nucleotide polymorphismRecently, in a genetic association study of rhegmatogenous retinal de-

tachment, an important cause of vision loss which potentially leads toblindness, a missense coding single-nucleotide polymorphism (E115A)in chromosome 1q21.3 was located in the Hox domain of the CerS2gene [72]; interestingly, inhibition of ceramide synthesis by myriocinslowed disease progression in a mouse model of retinitis pigmentosa[73]. Ceramides are known tomediate apoptosis ofmammalian photore-ceptors [74] and retinal pigment epithelial cell lines [75], and mutationsin the ceramide kinase-like (CERKL) gene cause autosomal recessive ret-initis pigmentosa [76,77], concomitant with decreased levels of SLs inCERKLnullmouse retina [78];moreover, inhibition of ceramide synthesisby FTY720 [79] protected retina from light-induced degeneration [80].Thus, C22–C24-SLs might be involved in the pathophysiology of retinitispigmentosa, which could be regulated by CerS2.

5.1.2. CerS3 mutationTwo studies have reported that mutations in the CerS3 gene lead to

skin ichthyosis [65,66]. In the first, the patient showedmoderate lamel-lar ichthyosis and hypohidrosis, and suffered from repeated bacterialand pityrosporum infections. This patient had only one variant, 43TNCin exon 4 of CerS3, resulting in mutation of the conserved residue,Trp15, to arginine, causing impaired activity and expression of CerS3.Amotif of ~30 residues in the N-terminal domain of CerS3, and particu-larly Trp15, are highly conserved, suggesting that this domain is impor-tant for protein function [65]. As a result of this mutation, levels ofC26:0-ceramides, and of ceramides containing ultra-long acyl chains,were also reduced in keratinocytes. In normal skin, CerS3 is expressedin the upper granular layer and in the stratum corneum, but wasfound in additional layers in the patient, along with earlier maturationof keratinocytes and epidermal barrier dysfunction.

Another study reported that additional CerS3 mutations, including amicrodeletion in chromosome 15q26.3 and a splice site mutation inCerS3 (c.609+1GNT), caused autosomal recessive congenital ichthyosis[66]. There was little CerS3 expression in the stratum granulosum andstratum corneum of the patients, and very-long chain ceramides de-creased by ~50%,with a small increase in C16–C20-ceramides. Thesemu-tations also caused abnormal epidermal differentiation. Both of thesestudies attest to the important roles that C26:0-ceramide generated byCerS3 play in epidermal differentiation, barrier integrity and the forma-tion of the cornified lipid envelope in humans. Moreover, the humanphenotype was similar to that observed in the mouse CerS3 knockout.

5.2. Cancer

The examples above, at least in the case of CerS3, show that a muta-tion in a CerS can be the direct cause of a humandisease. However, thereare a number of examples whereby changes in the expression levels orin the activity of one or other CerS correlates with human disease, al-though they are unlikely to be the direct cause of the disease. This is par-ticularly true in cancers.

One of the best-characterized examples is in head andneck squamouscell carcinoma, in which C18:0-ceramide levels are decreased, and ele-vating C18:0-ceramide levels, by transfecting with CerS1, inhibited cellgrowth by modulating the mitochondrial death pathway [81]. CerS1knockdown led to inhibition of caspase 3-like activation and cell deathby photodynamic therapy [82], whereas CerS1 overexpression enhancedthe growth-inhibitory effects of gemcitabine/doxorubicin by induction ofcaspase-3 activation [83]. In lymphovascular invasion and pathologicnodal metastasis, decreased C18:0-ceramide levels were related to theincidences of the cancer [47].

CerS levels are also altered in breast cancer. Increased levels of CerS2,4 and6mRNAexpressionwere observed, alongwith a decrease in sphin-gosine kinase 1 expression in estrogen receptor positive breast cancers



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[84,85]. A similar study demonstrated that total ceramide levels were el-evated in malignant breast cancer [86], and in particular, C16:0-, C24:1-and C24:0-ceramides were increased. Furthermore, up-regulation ofCerS4 and 6 led to inhibition of cell proliferation and induction of apopto-sis, and CerS2 up-regulation increased cell proliferation [41]. Anotherstudy showed that CerS2 expression was significantly lower in drug-resistant human breast cancer cells and low expression of CerS2 was as-sociated with poor prognosis in patients with breast cancer [87]. Finally,CerS2 enhanced chemosensitivity of breast cancer by counteracting theacidic tumor microenvironment through inhibiting the activity of theV-ATPase proton pump [87]. It appears that levels of CerS2, 4 and 6,and of sphingosine kinase, might be diagnostic or prognostic for the de-velopment of breast cancer, although studies usingmuch greater samplesizes are required before definitive conclusions can be reached.

CerS have also been implicated in the development of colon cancer,inasmuch as CerS6 expression is involved in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitivity in colon cells.TRAIL-sensitive SW480 cells contained higher levels of C16- and C18-ceramides and lower levels of C24-ceramides than TRAIL-resistantSW620 cells [88], TRAIL-resistant SW620 cells expressed lower levels ofCerS6 [89], and CerS6 overexpression in SW620 cells enhanced drug-induced CD95 activation and enhanced tumor cell death, whereas CerS6knockdown suppressed CD95 activation [90]. These studies support anegative correlation between colon tumor growth and CerS6 expression.

In summary, there appears to be a correlation between the extent ofproliferation and cell death in some cancers and levels of expression oractivity of specific CerS, which potentially might pave the way for ther-apies that regulate cancer cell growth via modulation of CerS activity.

5.3. Other diseases

5.3.1. CardiomyopathyCeramide acts as cardiotoxin in lipotoxic cardiomyopathy [91].

Inhibiting SL synthesis by myriocin improved systolic function and

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Currently-available CerS inhibitors. Note that none of the inhibitors are specific towards one o

CerS inhibitor IC50 (μM) Clinically useful features

Fumonisin B1 0.1 Alleviates gastric ulcersReduces folate stress-induce

Attenuate partial sciatic nertactile allodyniaPartially recovers insulin senAnti-apoptotic effect in a rodischemia and reperfusion inAttenuates allergic asthmatiinflammation in a Guinea piReduces SU5416-induced alin mouse and rat modelsInhibits amyloid beta-peptid

AAL-toxin 0.035–0.04

Australifungin 0.093 Displays broad-spectrum an

FTY720 6.4


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prolonged survival rates in dilated cardiomyopathy mouse models [91].Moreover, C14-ceramidewas required to generate hypertrophy in cardio-myocytes [92], suggesting a role for C14-ceramide in cardiac disease.

5.3.2. Multiple sclerosisIn an experimental autoimmune encephalomyelitis (EAE) model of

multiple sclerosis [93], C16-ceramide levelswere elevated in the lumbarspinal cord of EAE mice, which was accompanied by increased CerS6expression in monocytes/macrophages and astroglia [94]. Down-regulation of CerS6 reduced nitric oxide/TNF-α release whereas up-regulation of C16:0-ceramide had an opposite effect, suggesting thatCerS6 might play a critical role in the progression of multiple sclerosisby regulating nitric oxide and TNF-α synthesis.

5.3.3. DiabetesA number of studies have suggested that ceramide acts as a master

regulator of metabolism [95], and several studies have shown changesin levels of ceramides in diabetes. Accumulation of C18:0-, C22:0- andC24:1-dihydroceramides is involved in mediating glucolipotoxicity-induced β-cell apoptosis, and CerS inhibition blocks glucolipotoxicity-induced β-cell apoptosis [96]. In the CerS2 null mouse, hepatic insulinresistancewas observed, which affected hepatic insulin receptor activa-tion [58]. Hepatic cannabinoid-1 receptors were shown tomediate diet-induced insulin-resistance by increasing de novo synthesis of long-chain ceramides by activation of CerS1 and 6 [97].

6. CerS as putative therapeutic targets

Since CerS are involved in a number of human diseases, they couldpotentially act as novel therapeutic targets. Although a number of ge-neric CerS inhibitors are available (Table 4), none of them target specificCerS with a high degree of selectivity. Since the CerS are less specific to-wards the long chain base (see above), inhibitors would presumablyneed to be based on the high specificity of the CerS towards acyl CoAs.

r other CerS.

Cause of toxicity References

Failure of neural tube closure [99,101–104,122–130]d cell death Increases intestinal colonization by

pathogenic Escherichia coli in pigsve ligation-induced Induces hepatocarcinogenesis

sitivity in podocytesent model of splanchnicjuryc response and airwayg modelveolar septal destruction

e biogenesis[130,131]

tifungal activity [108,130]

Failure of neural tube closure [102,132]



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6.1. CerS inhibitors and activators

Fumonisin B1, a mycotoxin produced by Fusarium moniliforme, is byfar the best-characterized CerS inhibitor [98], and acts via competitive-like inhibition towards both sphinganine and acyl-CoA, with an IC50value of ~100 nM [99]. Fumonisin B1 contains an aminopentol backbonewith two hydroxyl groups esterified with tricarballylic acids [17,100].CerS inhibition occurs because the aminopentol moiety competes forbinding to LCBs and the dicarboxylic acid side chains interfere withacyl CoA binding [100]. However, the potential clinical use of fumonisinB1 is very limited due to its toxicity, which includes hepato-renal toxici-ty, pulmonary edema and encephalopathy [101–105]. TNF-α [105], reac-tive oxygen species [106] and N-acyl-fumonisin B1 formation [16] haveall been suggested as a mechanism of fumonisin toxicity. Other toxins,such as alternaria alternate toxin, produced by Alternari alternata [107],and australifungin, an antifungal agent from Sporormiella australis, arenon-sphingosine based CerS inhibitors [108]. Whereas CerS inhibitionby fumonisin B1 is relatively weak in vivo, possibly due to poor internal-ization, australifungin is a very potent CerS inhibitor; however, both thealpha-diketone and beta-ketoaldehyde functional groups limit its usedue to their high chemical reactivity [100]. The SL metabolic pathwayhas been targeted to develop antifungal drugs [109]; australifungin dis-plays broad-spectrum antifungal activity against human pathogenicfungi with minimum inhibitory concentrations between 0.015 and1.0 μg/ml [108].

In addition to the inhibitors mentioned above, all of which inhibitCerS in the high nM range, a number of other CerS inhibitors havebeen discovered, although most of these inhibit CerS in the mid μMrange. The first is FTY720, an FDA-approved drug for treatment of mul-tiple sclerosis [110], that upon its phosphorylation by sphingosine ki-nase, acts as an agonist of sphingosine 1-phosphate receptors [111].However, FTY720 also inhibits CerS by noncompetitive inhibition to-wards acyl CoAs and uncompetitive inhibition towards sphinganine[79]. Modulation of the FTY720 backbone, such as use of (oxy)deriva-tives with heterogeneous amine variations [112], is being attemptedby a variety of groups to attempt to increase the extent of inhibition.CerS activity can also be inhibited by the GlcCer synthase inhibitors,IV-231B and 11-threo-PDMPat 50 μM[113], but again, with no specific-ity towards the CerS. The development of CerS inhibitors specific for oneor other CerS is clearly of some clinical importance. For example, specificinhibitors of CerS1 or 5 might be clinically useful for improving hepaticinsulin resistance by decreasing long chain ceramides and elevatingvery-long chain ceramides [58,97], and specific CerS6 inhibitors couldbe clinically useful as therapeutic agents for multiple sclerosis [93].One general approach to developing such inhibitors might be via mod-ulation of the acid side chains of fumonisin B1, which act as analogs offatty acyl CoAs, or by the use of FTY720 analogs.

No activators of CerS are currently available, but the development ofsuch activators could be clinically useful in diseases such as head andneck squamous cell carcinoma [81], chemotherapy-resistant colon can-cer [90], and other diseases in which CerS activity is reduced. A numberof studies have shown that treatment of cultured cells results in elevat-ed ceramide formation [114–119], but there is no evidence that any ofthe drugs used act by direct activation of CerS; such evidence can onlybe obtained by in vitro analysis of CerS activity in the presence of puta-tive activators. Local administration of CerS activators into target tissueswould be optimal for therapy, and in principle, could be applied in thesame ways that ceramides are being applied, i.e., via nanoparticles andliposomes [120].

7. Summary

In this brief review, we have attempted to summarize currentknowledge about the state-of-art of research on the CerS, with an em-phasis on the role of CerS in human disease. While there is currentlyonly one example of a mutation in CerS leading to human disease


(CerS3), it is highly probable that mutations in additional CerS willsoon be discovered. Along with the role that ceramides and CerS playas secondary components in disease pathology, the development of in-hibitors and activators that target specific CerS becomes of paramountimportance. To date, such inhibitors are not available, but we predictthat the rapidly-growing pace of research into the CerS will lead to thedevelopment of such molecules in the near future.

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Acknowledgements

We thank Rotem Tidhar for help in making Fig. 1 and Dr. Shifra Ben-Dor (Bioinformatics and Biological Computing Unit, Weizmann Instituteof Science) for critical comments. Work in the Futerman Laboratory onthe CerS is supported by theMinerva Foundation, the Israel Science Foun-dation (0888/11) and theNational Institutes of Health (GM076217).W-J.Park was supported by the Basic Science Research Program through theNational Research Foundation of Korea (NRF) funded by the Ministry ofEducation, Science and Technology (2012R1A6A3A03038319). AnthonyH. Futerman is the Joseph Meyerhoff Professor of Biochemistry at theWeizmann Institute of Science.

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Ceramide synthases as potential targets for therapeutic intervention in human diseases