An Overview of Sphingolipid Metabolism: From Synthesis to Breakdown Part 2

Ceramide Kinase and Ceramide-1-Phosphate

Although ceramide is primarily converted into more complex sphingolipids in the Golgi, ceramide can also be phosphorylated to produce ceramide-1-phosphate (C1P). C1P is produced in the trans-Golgi and potentially the plasma membrane, by ceramide kinase (CERK). CERK, a member of the DAG kinase family, was originally identified based on its homology to sphingosine kinase. Unlike the sphingosine kinases, CERK only utilizes ceramide as a substrate and has no activity for sphingosine or DAG (Sugiura 2002). CERK activity is enhanced in the presence of calcium or magnesium and contains a putative calmodulin-like domain. In addition, ceramide kinase has specificity for sphingosine containing ceramides since it has very low activity against dihydroceramide and phytoceramide species.59, 60 Among the ceramide species it recognizes, CERK prefers ceramide species with acyl chain lengths greater than 12 carbons long, however, no preference was observed for the degree of saturation.60 The measurement of C1P levels in A549 lung adenocarcinoma cells revealed an enrichment of C1P species containing acyl chain lengths of C16, C18 and C20 relative to their respective ceramide species.61 The enrichment for particular ceramide species for C1P was suggested to be due to specific delivery of ceramides to the trans-Golgi by CERT, a lipid transport protein which is biased for ceramide species with acyl chain lengths less than 22 carbons.62 Knockdown of CERT using RNA interference led to a decrease in C1P levels in A549 cells. A separate study using a pharmacological approach for CERT inhibition, found that inhibiting CERT had no effect on C1P production, but still inhibited sphingomyelin production.61,63 It is unclear if these differences were due to intrinsic differences in the cell types studied (human lung versus mouse macrophage) or due to nonspecific effects of either approach. Future studies are needed to resolve this discrepancy.

CERK displays significant homology to other DAG kinases, however it also contains a N-terminal myristoylation site and a pleckstrin homology (PH) domain.64 The PH domain targets CERK to PIP2 containing membranes, but is also necessary for enzymatic activity. Several studies have shown that CERK is localized to the trans-Golgi in a PH dependent manner. C1P generated in the Golgi can act as a docking site for cytosolic PLA2 and enhances arachidonic acid release. In addition, CERK translocates to the plasma membrane in response to osmotic swelling, an insult that enhances PIP2 on the plasma membrane. Translocation of CERK to the plasma membrane was shown to also be dependent on its PH domain.

The generation of a Cerk-/- mouse has provided some clues to CERK function in vivo. Cerk-/-mice are fully viable and show no gross phenotypic changes suggesting that CERK is not essential for development. Lipid analysis of serum from Cerk-/- revealed greatly elevated ceramides, but decreased dihydroceramides suggesting that CERK contributes significantly to ceramide metabolism in the serum.59 Upon closer examination, Cerk-/- mice were found to have a normal level of C1P in their brains, despite a lack of CERK activity, suggesting that C1P can be produced through a mechanism independent of CERK.65 Behavioral testing of the mice did, however, show abnormal emotional behavior based on an increase in ambulation and defecation frequencies in an open field test.65 More recently, the Cerk-/- mice were found to have significant neutropenia under basal conditions. When these mice were challenged with S. pneumoniae they succumbed to lethal pneumonia earlier than wildtype mice and had a higher bacterial burden in their lungs.66

A CERK homologue formerly named retinitis pigmentosa 26, RP26, but recently renamed CERKL, or CERK-like, has been identified. Since this gene has been implicated in a human form of retinitis pigmentosa it was initially suspected that C1P might play an essential role in retinal biology. Initial expression studies of CERKL expressed in cells failed to detect any CERK activity drawing into question if CERKL was an actual ceramide kinase.67,68 After generation of a CERKL knockout mouse, it was determined that Cerkt– mice had no alterations in retinal C1P, ceramide, or CERK activity.59 CERK-/- mice, on the other hand, had 80% less C1P levels and elevated ceramides suggesting that CERK is the major enzyme responsible for C1P in the retina.59 Taken together, it is unlikely that CERKL is a true ceramide kinase, but may have a separate function which is essential for retinal cells. Also, it is interesting to note that this study found that the retina of Cerk-/- mice had greatly reduced C1P levels, whereas previous studies have shown that whole brain C1P in these mice was unchanged.59,65 Future studies will help address the contribution of CERK to C1P levels and potentially identify other enzymes involved in C1P production.

C1P levels are regulated both by its synthesis through CERK, but also by its dephosphorylation back into ceramide. It is unclear how ceramide-1-phosphate is dephosphorylated, however, several groups have reported C1P phosphatase activity in plasma membrane fractions in both the liver and brain.69-71 C1P has been shown to traffic through the secretory pathway to reach the plasma membrane where it could potentially be dephosphorylated by C1P phosphatases.63 Also, C1P appears to be a substrate for nonspecific lipid phosphatases of the LPP family.

Catabolizing Complex Sphingolipids and Sphingomyelins into Ceramide

There is a significant tradeoff between the ability of an organism to produce a novel advantageous lipid and the potential that the organism is incapable of catabolizing the same lipid, resulting in accumulation of a lipid product in cells or tissues. Lipids pose an additional problem in that they can not be excreted as readily as other more hydrophilic molecules and therefore tend to accumulate within cells when they can not be destroyed. As such, it is not surprising that for every enzyme capable of generating a specific sphingolipid, there exists an ‘opposing’ enzyme capable of breaking down the generated product. Indeed, the mutation of specific catabolic enzymes is the general principle behind lipid storage diseases and defects in sphingolipid catabolizing enzymes are responsible for a significant number of these diseases. Due to the limited scope of this topic, the reader is referred to several recent reviews on lipid storage diseases for more expansive information on the variety of lipid storage diseases due to glycosphingolipid enzyme mutations.72,73 Suffice it to say that many lysosomal hydrolases are necessary for the coordinated breakdown of complex glycosphingolipids and the absence of any single one of these results in the accumulation of its respective substrate.

Sphingomyelin is the most abundant complex sphingolipid in human cells. Therefore, coordinated breakdown of sphingomyelin is an essential part of membrane homeostasis. Breakdown of sphingomyelin occurs through the hydrolysis of the phosphocholine headgroups by the sphingomyelinase family. The direct result of sphingomyelin hydrolysis is the production of ceramide and free phosphocholine. The mammalian sphingomyelinases fall into three major categories based upon their pH optimum: acid sphingomyelinase, alkaline sphingomyelinase and the neutral sphingomyelinases. Although all three forms of sphingomyelinases catalyze a similar reaction, these three groups of enzymes are evolutionarily unrelated and have different subcellular distributions. Alkaline sphingomyelinase, which is exclusively expressed in the intestine and liver, plays a role in the digestion of dietary sphingomyelin and will not be discussed further in this section.74 Acid sphingomyelinase and neutral sphingomyelinase are ubiquitously expressed and serve as the major regulators of SM catabolism in most tissues and will be discussed in more detail.

Acid sphingomyelinase (ASMase) was the first sphingomyelinase to be characterized in mammalian cells. ASMase is predominantly a lysosomal protein which metabolizes sphingomyelin present on endosomal membranes. Lysosomal ASMase becomes N-glycosylated on at least six residues within the ER which stabilizes the enzyme structure and provides protection from proteolysis within the lysosomes. ASMase also becomes mannose-6 phosphorylated within the Golgi which directs it into the lysosomal compartment where it is most active.75 The ability of ASMase to reach the lysosomal compartment is essential for its ability to catabolize sphingomyelin.

ASMase is also secreted into the extracellular space (where it is often referred to as secretory SMase) where it has access to sphingomyelin-containing lipoproteins which are abundant in the plasma.76 In addition, secretory ASMase can metabolize outer leaflet SM on the plasma membrane. Secretory ASMase, unlike the lysosomal form, requires zinc for sphingomyelinase activity. It is unclear how the secretion of ASMase is regulated, but it appears to be through the constitutive secretory pathway. Moreover, the function of secretory ASMase is still unclear, but is thought to play a role in reducing plasma SM content and may play a role in cellular stress responses by generating plasma membrane localized ceramide with a specific signaling role.

Insights into the significance of the acid sphingomyelinase protein, encoded by the gene SMPD1, come from a human lysosomal storage disorder Niemann Pick Disease Types A (NPD A) and B (NPD B).77 Complete absence of a functional ASMase gene product results in NPD A which is characterized by a progressive neurodegenerative disease with psychomotor retardation, retinal cherry red spots, hepatosplenomegaly, lung disease and premature death. Patients afflicted with NPD A usually do not live past the age of three. A mouse model of NPD A, Smpd1-/-, exhibits growth defects similar to the human disease and also dies prematurely around 4 months of age.78 NPD B, on the other hand, is a less severe form of the disease that lacks neuronopathic symptoms, however, hepatosplenomegaly and lung disease still occur. Fortunately, individuals afflicted with NPD B are able to survive into adulthood. The NPD B phenotype has been mimicked in mice by fusing a Smpdl transgene to the LAMP1 gene.79 In these mice there is complete absence of secretory SMase, but low level lysosomal ASMase activity. Future studies will dissect which symptoms of NPD are likely due to a lack of secretory ASMase function and which symptoms are associated with lysosomal ASMase function, thereby elucidating further some of the specific functions of each form of the enzyme.

Within the past decade, three different mammalian neutral sphingomyelinase (NSMase) genes have been identified, SMPD2, SMPD3 and SMPD4, although, the first NSMase gene discovered, SMPD2, is not likely to function as a SMase, but rather as a lyso-PAF phospholipase C.80 To further muddy the point, mice lacking the Smpd2 gene have a decreased tissue SMase activity, but have no alteration in tissue SM levels.81

The best characterized NSMase to date is NSmase 2. NSMase 2 contains two highly hydrophobic domains, that may function as membrane anchors but not full transmembrane domains.82 Unusually, N-SMase2 is thought to have its catalytic site facing the cytosolic leaflet of either the Golgi or plasma membrane. This orientation for a SMase is unusual since SM is thought to be relatively excluded from the cytosolic leaflet.82 Interestingly, NSMase2 localizes to the Golgi under subconfluent conditions, however upon reaching confluence, NSMase2 translocates to the plasma membrane.83 It was later shown that plasma membrane association is highly dependent upon its palmitoylation on multiple cysteine residues.82

NSMase2 overexpression, in the absence of a specific stimulus causes degradation ofsphingomy-elin into ceramide with a preference for C24:0 and C24:1 species.83 Generation of C24 and C24:1 ceramide during confluence dependent growth arrest was dependent on NSMase 2 suggesting that generation of specific ceramide species could have specific effects on growth arrest.83 These results also suggest that NSMase2 either has specificity for very long chain sphingomyelin species or that very long chain sphingomyelins are enriched in the inner leaflet of the plasma membrane. Since most sphingomyelin species are thought to be localized to the outer leaflet of the plasma membrane or the luminal side of the Golgi, it is a mystery how NSMase2 with an inner leaflet catalytic site could access its substrate. The condundrum of how NSMase2 reaches its substrate undoubtedly puts into question some existing paradigms about sphingomyelin localization in membrane leaflets.

The generation of a Smpd3-/- mice has provided some insights into the physiological role of NSMase 2.84 Smpd3-/- mice display severe growth retardation, organ hypoplasia, delayed puberty and skeletal defects. Upon further examination, a combined pituitary hormone deficiency was discovered in these mice and a significant decrease in serum IGF-1, TSH and GnRH was found. Due to the complex interactions between the pituitary hormones and target tissues it is difficult to dissect which symptoms are due to hormone deficiency and which defects are due to primary organ defects. Some insight into this was provided by the generation of a chondrocyte specific SMPD3 expression mouse. When the chondrocyte specific SMPD3 mouse was crossed with the Smpd3-/- mouse, there was an absence of skeletal defects, suggesting that chondrocyte specific NSMase2 activity is essential for skeletal development.85 Despite the dramatic pathology observed with the Smpd3-/- mouse, it is still difficult to understand how the NSMase2 knockout phenotype correlates with an intracellular role of NSMase 2 in sphingomyelin generation. Further characterizing the cellular dysfunction in the Smpd3-/- mouse in parallel with further characterizing N-SMase 2 in vitro will shed light on a murky area of sphingolipid biology and provide new insights into cell biology as a whole.

Finally, a third NSMase, NSMase3 has recently been identified which is encoded by the SMPD4 gene.86 Interestingly, NSMase3 is localized to the ER and possibly the Golgi. It was found to contain at least one transmembrane domain and potentially more along with an ER retention signal.87 NSMase3 is predominantly expressed in skeletal and cardiac muscle with minor expression in many other tissues. It will be interesting to see how an ER localized SMase can affect sphingolipid metabolism since SM is not thought to be present in the ER compartment.

The Catabolism of Ceramides and the Final Common Breakdown Pathway

Just as a few sphingolipid precursors are generated to produce hundreds of different sphin-golipids, all sphingolipids are eventually catabolized to ceramide, sphingosine and finally, sphingosine-1-phosphate. The deacylation of ceramide species is achieved through the family of enzymes known as ceramidases. These ceramidases have organelle specific expression and may have specificity for different forms of ceramide to bias a cell towards the generation of complex sphin-golipids with specific sphingoid bases. Organelle specific expression also allows for the possibility to serve as negative regulators of organelle specific ceramide signaling. After ceramide is deacylated into sphingosine, the conversion of sphingosine to sphingosine-1-phosphate is achieved through one of two sphingosine kinases localized in the cytosol or peripherally associated with specific membrane compartments. In the final step of sphingolipid breakdown, sphingosine-1-phosphate is degraded by the enzyme sphingosine-1-phosphate lyase in the ER to produce hexadecenal and phosphoethanolamine.

Acid, Neutral and Alkaline Ceramidases

The ceramidases, like many other sphingolipid enzymes, have been classified biochemically according to their pH optima. Acid ceramidase (AC), as its name suggests, is a lysosomal enzyme which deacylates ceramide species produced from the degradation of plasma membrane sphingolipids. AC, encoded by the ASAH1 gene, is a member of the N-terminal nucleophile (Ntn) hydrolase superfamily. Members of the Ntn family are characterized by their ability to undergo autoproteolytic cleavage through cysteine dependent proteolysis. Interestingly, this processing was shown to be accelerated at pH 4.5 when compared to neutral pH. This pH dependent maturation is likely a mechanism that has evolved to prevent premature activation of AC prior to it reaching the lysosomal compartment. AC activity was shown to be higher against C12 and C14 ceramide species compared with C6 or C18 ceramide species.88 Although it has been reported that AC has the greatest activity against medium and long-chain ceramide species, it is difficult to imagine how very long-chain ceramides would be processed in a compartment where AC is the only known ceramidase.88 Therefore, it is likely that very long-chain ceramide containing sphingolipids are either excluded from the endolysosomal compartment or that AC is able to degrade these very long chain ceramides albeit at a slower rate or with the aid of an accessory protein.

The significance of AC in mammalian systems is reinforced by its role in the human lipid storage disease Farber lipogranulomatosis. Farber disease is an autosomal recessive disorder due to a dysfunctional AC gene product. Farber’s disease is characterized by early onset arthritis, swollen lymph nodes, psychomotor difficulties and vocal cord pathology. Complete absence of AC expression in a mouse model results in embryonic lethality at a very early stage in development.89 Mice heterozygous for AC develop a progressive lipid storage disease due to ceramide accumulation in the liver, skin, lungs and bones. The striking phenotype of the AC heterozygous mice raises questions about its implications for human disease. One could imagine that a heterozygous defect in AC within a human population may lead to a progressive lipid storage disorder that may only express itself with advanced age. Answers to questions like these will most likely be revealed when human genome sequencing becomes more widespread.

Neutral ceramidase, the most active ceramidase at neutral pH is encoded by the ASAH2 gene. Neutral ceramidase (NC) is synthesized through the secretory pathway as a Type II integral membrane protein. NC can be cleaved at its N-terminus to produce a soluble protein that peripherally associates with the outer leaflet ofthe plasma membrane. NC contains mucin box domains which are highly O-glycosylated and are necessary for plasma membrane association.90 NC associated with the plasma membrane is an important regulator of sphingosine and sphingosine-1-phosphate (S1P) production and for S1P release.91 In addition, NC is highly expressed in the intestinal epithelium and contributes to the digestion of dietary sphingolipids.92,93 Asab2~’~ mice showed an inability to metabolize dietary ceramides. Although, sphingosine and ceramide levels were normal in the brain, liver and kidney of Asab2-/- mice, the intestines had a significantly increased C16:0 ceramide content but a reduced sphingosine content. Due to an absence of visible pathology in the Asab2~’~ mice, it is unclear what role NC plays in nonintestinal tissues, if any.92 Future studies with the Asab2~’~ mice may reveal subtle or previously uncharacterized, but important functions for this gene under specific stresses.

The alkaline ceramidases (ACERs) contain three separate family members: alkaline cerami-dases 1, 2 and 3 are encoded by the ASAH3,94 ASAH3L95 and PHCA96 genes respectively, which share significant homology to one another. These ACERs differ in subcellular localizations and substrate specificity although they have alkaline pH optima for their in vitro activity and are activated by calcium ion in vitro.97 ACER1 is mainly expressed in the epidermis, whereas ACER2 and ACER3 are expressed in various tissues. ACER1 has multiple putative transmembrane domains and is localized to the ER.94,98 Interestingly, ACER1 has marked substrate specificity for C24 and C24:1 ceramides, but has no activity against dihydroceramides or phytoceramides.94,98 This highly restricted substrate specificity may prevent phytoceramide species from being degraded because the skin is one of the limited tissues that is enriched in these types of ceramides.

ACER2, also referred to as Golgi alkaline ceramidase, is highly expressed in the placenta, but its low expression can be detected in most tissues. Like its homologue ACER1, ACER2 has several putative transmembrane domains, however, ACER2 is localized to the Golgi complex. ACER2 has a less restricted substrate specificity since it also metabolizes other long-chain ceramides C16, C18 and C20 ceramide species and long-chain dihydroceramides and phytoceramides with an unsatu-rated acyl chain, in addition to C24 and C24:1 species (Cungui Mao personal communication). ACER2 requires calcium ions but not other cations for its activity.

ACER3 was previously called phytoceramidase because it was found to have higher in vitro activity towards the artificial fluorescent phytoceramide D-ribo-C12-NBD-phytoceramide, than towards NBD-ceramide or NBD-dihydroceramide.96 Mao et al recently found that ACER3 only catalyzes the hydrolysis of natural phytoceramide, dihydroceramide and ceramides carrying an unsaturated fatty acid (< C20) (Cungui Mao personal communication). ACER3 appears to be the only ceramidase identified in mammals which has a preference for phytoceramide species. Its tissue expression is widespread, but, like ACER2, is highly expressed in the placenta. ACER3 is localized to both the ER and Golgi complex, with a C-terminal ER retention sequence.96 Interestingly, its activity is inhibited by sphingosine, but not by dihydrosphingosine or phy-tosphingosine. The significance of this mammalian ceramidase remains to be seen, but its unusual specificity for phytoceramide raises questions about the unique functions of phytoceramide containing sphingolipids.

Although investigation has begun to classify and characterize the different biochemical properties ofthe ceramidases, much still remains to be done to define the specific functions ofeach alkaline ceramidase. The generation of the three alkaline ceramidase knockout mice will undoubtedly shed further light on this subject.

Sphingosine-1-Phosphate and Sphingosine Kinases 1 and 2

The two sphingosine kinases (SK), SK1 and SK2 are members of the DAG kinase family.99 Both enzymes utilize ATP to phosphorylate the C-1 hydroxy group of free sphingosine, dihydrosphingosine, or, in the case of SK2, also phytosphingosine. Both sphingosine kinases are cytosolic enzymes that peripherally associate with membranes. Regulation of sphingosine kinase localization within the cell is thought to be the primary mode by which these enzymes acutely affect sphingolipid metabolism since only modest changes in their activity can be detected after stimulation by various agonists.100,101 In addition, SK enzymes are regulated transcriptionally by a variety of stimuli,102-104 Although SK1 and SK2 both catalyze the same reaction, they have slight differences in their substrate specificities and have distinct, but overlapping, subcellular localizations which determine their effects on specific sphingolipid compartments within cells.

Sphingosine kinase 1 (SK1) is a cytosolic enzyme, but it can associate with the plasma membrane, move into the nucleus and even be secreted from cells.100,105-108 The first major insight into the regulation of SK1 activity first came when it was shown that SK1 translocates from the cytoplasm to the plasma membrane in response to phorbol ester treatment.100 Shortly after this, translocation to the plasma membrane was shown to be phosphorylation dependent and that ERK2 was the kinase responsible for this phosphorylation.101 Later work showed that phosphorylation of SK1 lead to its enhanced affinity to anionic phospholipids such as PS, PI and PA which are in high abundance on the inner leaflet of the plasma membrane.109 The metabolic significance of SK1 translocation to the plasma membrane was shown to be enhancement of S1P production and extracellular release of S1P.100,110 This agonist induced translocation of SK1 through phosphorylation by ERK2, has become the general paradigm for S1P signaling and receptor activation in response to various agonists. It is worth noting that many different growth factors converge on SK1 as an intracellular target for downstream signaling. These include, but are not limited to, PDGF, VEGF, NGF, EGF, Insulin and IGF-1. Activation of SK1 was shown to be necessary for the proliferative effects of many different growth factors.111-116 Whether or not all of these growth factors act through the same intracellular signaling pathway to activate SK1 remains to be tested. The reader is referred to several good reviews on SK1 for more detailed information on its signaling role in proliferation and survival.35,117,118

Although phosphorylation dependent translocation of SK1 to the plasma membrane has been the best defined mechanism by which SK1 activity is regulated, SK1 also traffics through the nucleus. SK1 was shown to have two putative nuclear export sequences (NES) which are necessary for SK1 to leave the nucleus.107 It is unclear how SK1 introduction into the nucleus is regulated, but deletion of the two nuclear export sequences present on SK1, lead to nuclear accumulation of SK1. The effect nuclear trafficking of SK1 has on sphingolipid metabolism is unclear, but attachment of a nuclear localization sequence on SK1 inhibited cellular proliferation through an unknown mechanism.119

In addition, SK1 has been shown to be secreted from endothelial cells, however, the physiological significance of this is not clear. An interesting discovery will be the route by which an exclusively cytosolic protein gets secreted from endothelial cells. Analysis of SK in endothelial cells showed that secretion was constitutive and not regulated by agonist stimulation.106 It has been suggested that extracellular SK can generate S1P in the extracellular space following coordinated hydrolysis of plasma SM by secretory SMase and NCDase.120 Some evidence for SK secretion occurring in vivo comes from Venkataraman et al. A significant amount of soluble SK activity was found in the serum of wildtype, but not SphK1-/- mice suggesting that SK1 is indeed released into the blood of mice.121 Once again, the physiological significance of SK1 release remains to be determined.

In addition to its effects on promoting proliferation, SK1 has plays an important role in survival and resistance to chemotherapy.122 Overexpression of SK1 correlates with a resistance to chemotherapeutic agents.123 In addition, overexpressing SK1 in sensitive cells can induce resistance to chemotherapy.105 Importantly, it has been shown that SK1 degradation is a downstream event in the DNA damage response.124 Apoptosis inducing agents, such as TNF-a or various DNA damaging agents lead to degradation of SK1 through a Cathepsin B dependent mechanism.125 In vitro characterization of SK1 degradation has shown that cathepsin B degrades SK1 through a stepwise cleavage, first at Histidine 122 followed by cleavage at Arginine 199.126 How DNA damaging agents or death inducing ligands, such as TNF-a, induce degradation of cytosolic SK1 through lysosomal localized cathepsin B is not yet clear. Future studies will better define how SK1 contributes to survival and how its degradation is involved in apoptosis.

Sphingosine kinase 2, the lesser studied isoform of the two, is predominantly localized in the nucleus or perinuclear region of a cell. SK2 has broader substrate specificity than SK1 and is able to phosphorylate phytosphingosine as well as sphingosine and dihydrosphingosine. Early reports on SK2 function suggest that it enhances apoptotic responses and can induce apoptosis through overexpression.127 This is in contrast to SK1, which enhances survival of cells when overexpressed and allows for resistance to apoptosis inducing agents.122 Due to its perinuclear localization, SK2 is thought to produce phosphate bases distal from the plasma membrane, where ABC transporters are less likely to export them into the extracellular space.128 Another explanation for their disparate functions comes from a recent study suggesting that SK2 uniquely couples to sphingosine phosphate phosphatase 1 (SPP1) which generates free sphingosine for ceramide synthesis.129 Interestingly, predominantly C16, C18 and C20 ceramide species were produced through this pathway, suggesting that the recycling pathway through SK2 and SPP1 may couple to specific ceramide synthases. Importantly, SK1 was found not to have the same function in enhancing sphingosine recycling into ceramide. Therefore, SK2 may have a distinct function from SK1 by having an enhanced ability to recycle sphingoid bases for ceramide synthesis. The structural basis for this difference has not yet been characterized, but will help elucidate how two enzymes with the same catalytic activity can have dramatic differences on cell biology.127

Insight into the physiological role of S1P was hoped to be gained through generation of sphin-gosine kinase 1 knockout mice, however, in the absence of stresses they displayed no obvious phe-notypic abnormalities.130 In contrast, SpbK1-/- mice develop less intestinal adenomas when crossed with the Apc^,n/* mice, a model of intestinal adenocarcinoma.131 SpbK1-/- mice also develop less adenocarcinomas when treated with DSS/AOM.132 Moreover, SpbK1-/- mice are less susceptible to DSS induced colitis and have a marked decrease in intestinal inflammation in that model.133

Sphingosine kinase 2 knockout mice have also been generated and these mice too do not display any obvious abnormalities.134 Crossing of SpbK1-/- and SpbK2-/- mice revealed that complete absence of SK activity and therefore S1P, results in embryonic lethality due to improper neural and vascular development.134 These defects are likely to be due to a lack of S1P and not due to sphingosine accumulation since sphingosine levels were actually below wildtype levels in double knockout embryos. In addition, the vascular defects observed in the double knockout mice resembled previously observed defects in S1P1 receptor knockout mice, suggesting that S1P is absolutely necessary for vascular development.134,135 Finally, female SpbK1-/-, SpbK2 +/- mice are infertile due to defects in decidualization.136 Together these studies suggest that SK1 and SK2 have redundant functions during development since neither SK knockout displays any developmental defects alone, but display severe defects when both enzymes are absent. On the other hand, each SK isozyme may have unique functions in mature tissues since SpbK1-/- mice have specific defects in inflammatory responses.133

Lipid Phosphate Phosphatases, S1P Phosphatases and the Salvage Pathway

Sphingosine-1-phosphate can be dephosphorylated at the cell surface by a family of broad specificity lipid phosphate phosphatases, LPP1-3. LPPs have six transmembrane domains and have their catalytic sites facing the extracellular space. The LPP family is important for sphingolipid metabolism because they are thought to be the primary mechanism by which extracellular S1P signaling is attenuated.137,138 Overexpression of specific LPPs reduces S1P-dependent signaling events.139,140 LPP effects on S1P metabolism is complicated by the fact that LPPs also affect phosphatidic acid (PA) levels which have been shown to regulate SK1 localization in cells.138,141 In addition, S1P may require LPP dependent dephosphorylation prior to its uptake by cells, although a separate mechanism for uptake requiring the CFTR protein has also been proposed.142 For additional information on LPPs, the reader is referred to some recent reviews.138,143

In addition to LPPs, cytosolic S1P can be dephosphorylated at the ER by S1P specific phosphatases, SPP1 and SPP2.144-146 SPP1 and perhaps SPP2, plays a role in regulating the reintroduc-tion of sphingoid bases into ceramide species at the ER.129,147 Distantly related to LPPs, SPP1 and 2 each contain eight putative transmembrane domains.145,148,149 Interestingly, overexpression of SPP1 results in an increase in ceramide accumulation suggesting that dephosphorylation of S1P is a rate limiting step in the salvage pathway.129,145,150 This increase in ceramide can be exacerbated further by the addition of extracellular S1P. Therefore, regulation of SPP1 levels can change the metabolic fate of S1P to be predominantly recycled into ceramide. This has been shown to have dramatic effects on the biological response of cells to S1P.150 Recently, SPP1 mediated conversion to ceramide was shown to be enhanced by overexpression of SK2, suggesting that SK2 and SPP1 work coordinately to increase sphingosine salvage.129 In addition, higher expression of SPP1 reduced extracellular release of S1P, suggesting that SPP1 can negatively regulate extracellular signaling of S1P.149

A second sphingosine phosphate phosphatase, SPP2, was identified based on homology to SPP1. Both SPP isoforms are expressed ubiquitously with high expression in the kidney. On the other hand, SPP1 is highly expressed in the placenta whereas SPP2 is highly expressed in the heart.146 More recently, SPP2 was shown to be upregulated during inflammatory responses, however, it is unclear what effect this has on sphingolipid metabolism.151 It remains to be seen if SPP2 plays an important role in regulating the sphingolipid recycling pathway like SPP1.

S1P Lyase in the Removal of Sphingoid Bases

Sphingosine-1-phosphate lyase (SPL) is the enzyme responsible for the conversion ofphospho-rylated sphingoid bases to hexadecenal and phosphoethanolamine, thereby serving as the final step in sphingolipid degradation. SPL is a single pass transmembrane protein, displaying Type I topology and is exclusively localized to the ER.152 The catalytic site of SPL faces the cytosolic surface of the ER where it has access to cytosolically produced S1P. Since SPL is exclusively localized to the ER, all sphingoid phosphate bases must reach the ER for their final degradation by SPL. SPL has broad substrate specificity since it can utilize sphingosine-1-phosphate, dihydrosphingosine-1-phosphate and phytosphingoinse-1-phosphate as substrates and therefore is able to catabolize all sphingoid bases found in mammals.153 Like SPT, SPL is dependent on pyridoxal 5′-phosphate (PLP) as a cofactor for enzymatic activity, making both the initial and the final steps in the sphingolipid pathway PLP dependent. The relationship between symptoms associated with vitamin B6 deficiency, the dietary precursor to PLP and alterations in sphingolipid metabolism are unclear, but will be important to assess since inhibition of SPT or S1P lyase could result in demyelination and altered immune function respectively.8,154

SPL has a wide tissue distribution with its highest expression in the thymus and intestines and its lowest expression in the brain and skeletal muscle.152 In addition, no SPL activity can be detected in platelets or red blood cells since these cells lack ER membranes.152 Immunohistochemical staining for SPL within the intestine revealed high expression on differentiated enterocytes, but low expression in the intestinal cypt cells.155 In addition, intestinal adenomas from ApcMm/* mice showed a marked decrease in SPL expression compared with adjacent normal mucosa.131 Together these data suggest that SPL upregulation is part of the normal differentiation of enterocytes. SPL expression in the thymus was also assessed and was localized almost exclusively to thymic epithelium with low expression in lymphocytes. Thymic expression of SPL seems to be essential for proper lymphocyte trafficking between the blood, primary lymphoid tissue and secondary lymphoid tissue due its role in establishing S1P gradients between the different trafficking compartments.

Several studies have identified S1P lyase as an essential gene for development in Drosopbila melanogaster, Caenorbibitis elegans and Dictyostelium discoideum}’,',’l',1 Recently, the phenotype of the S1P lyase knockout mouse, Sgpl1-/-, was published.158 These SPL defective mice display an inability to gain weight and premature death by eight weeks of age. These mice also displayed an array ofdefects which closely resembled those seen with the PDGF receptor, Pdgfra-/- and Pdgfrb—, mutant mice. Some of these defects include vascular hemorrhaging, defective glomeruli formation and skeletal defects. It is unclear if Sgpl1-/- mice had defects in any other systems since the study was interested only in PDGF dependent phenotypes.158 Regardless, this study provides clear evidence that SPL plays an essential role in the development of many tissues in mammals. Future studies will help identify the role of SPL in adult tissues and assess its potential as a therapeutic target for the treatment of disorders ranging from immunological disorders to neoplasias.


Sphingolipids are a diverse group of lipids which serve a variety of functions in both mammalian development and physiology. Only a few of these functions have been highlighted in the topic, but the scope of sphingolipid biology is vast. New functions for sphingolipids in mammalian physiology continue to be discovered each year and will likely increase as our understanding of sphingolipid biology in whole organisms improves.

It is clear that sphingolipids play an essential role in mammalian systems, therefore, it is essential to understand how sphingolipids are synthesized and degraded to maintain their functional levels at both an organismal and cellular level. Thanks to the hard work of many pioneers in the sphingolipid field, the biochemical characterization of many sphingolipid enzymes was laid out before any genes were identified. Aided by the advancement of recombinant DNA technology and the human genome project, significant progress has been made to clone and characterize the enzymes responsible for sphingolipid metabolism. At least one gene and in most cases two or more, has been cloned for each of the known enzymatic steps required from the condensation of serine and palmitoyl CoA by SPT to the production of hexadecenal and phosphoethanolamine by S1P lyase. Although it is easy to assume that we have a near complete list of the enzymes involved in sphingolipid metabolism, new isoforms of these enzymes are still being discovered and validated. While having a list of enzymes is comforting, our understanding of their regulation is still lacking. We still have a lot of work to understand how the different sphingolipid enzymes work in concert to determine the sphingolipid composition of the plasma membrane and subcellular organelles. In addition, the breakthrough discoveries of the sphingolipid transfer proteins CERT and FAPP2 have provided a new paradigm for nonvesicular sphingolipid trafficking. These proteins are likely to only be the first members of a class of sphingolipid transfer proteins which regulate the trafficking of sphingolipids to specific compartments within cells.

Although this topic aimed to provide an abbreviated overview of our current understanding of sphingolipid metabolism in mammalian systems, it also highlights some areas of lipid biology that are poorly understood. It is a healthy scientific practice to regularly reflect on the progress that has been achieved within a field, both, to bring to light significant faults or assumptions we have about it, but also for the sense of awe one gets about our collective accomplishment.

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