1. Structure and Biosynthesis in Animals
Ceramide-1-phosphate, a sphingoid structural analogue of phosphatidic acid, is one of the metabolites in the 'sphingomyelin cycle'. It is a bioactive phosphosphingolipid involved in regulating vital cell functions, including cell growth and survival, often acting in opposition to the effects of another sphingolipid mediator, i.e. ceramides. Ceramide-1-phosphate is present in animal tissues at a level comparable to that of sphingosine-1-phosphate (0.5-1μM in peripheral blood), and it is presumed to be located at the cytosolic leaflet of cellular membranes. Relatively high concentration of palmitoylated (C16) ceramide-1-phosphate have been observed in macrophages, mast cells and neutrophils.
In animal tissues, ceramide-1-phosphate is formed from ceramide by the action of a specific ceramide kinase, which is related to but distinct from the sphingosine kinases that synthesise sphingosine-1-phosphate. There is evidence that the ceramide precursor is derived primarily from sphingomyelin by the action of sphingomyelinases and only at trace levels from other sphingolipids. A specific pool of ceramide containing 16:0 and 18:0 fatty acid components is transported to the site of synthesis by the ceramide transport protein (CERT) for conversion to ceramide-1-phosphate by ceramide kinase (CERK), the only known biosynthetic route to this lipid in mammalian cells.
CERK is associated mainly with membranes, especially the trans-Golgi network at the cytosolic face, although it has also been detected in the cytosol, nucleus, perinuclear membranes and plasma membrane. It utilizes ATP as the phosphate donor, it has an absolute requirement for calcium ions, and it can be stimulated by interleukin 1-beta (IL-1β). The enzyme is optimally active at neutral pH, and regulation is by phosphorylation/dephosphorylation processes. It was first detected in brain synaptic vesicles and in human leukaemia (HL 60) cells, but it has since been found in many other tissues, and especially brain, heart, skeletal muscle, kidney, and liver. CERK is specific for natural ceramides with the erythro configuration and a 4,5-trans double bond in the base component and esterified to long-chain fatty acids (C16 especially). It has a calmodulin-binding motif, but an interesting relationship to glycerophospholipid metabolism is evident in that a molecule of phosphatidylinositol 4,5-bisphosphate appears to bind selectively to CERK via a Pleckstrin homology domain. This lipid may also direct the enzyme to particular membranes within the cell.
A membrane-bound transport protein, i.e. ceramide-1-phosphate transfer protein or CPTP (once thought to be a glycolipid transfer protein, GLTPD1 or CPTP), is essential for the biosynthesis of ceramide-1-phosphate. It appears to maintain a constant level of the lipid in the Golgi membrane and transfers it by a non-vesicular mechanism to the plasma membrane or other cellular compartments as required. The protein is mainly present in the cytosol, but is also found in association with nuclear membranes, and at the plasma membrane, it may be recognized by an interaction with phosphatidylserine.
Other biosynthetic routes to this lipid must exist, but have yet to be demonstrated experimentally, as mice in which the CERK enzyme has been deleted have normal levels of the metabolite. One possibility is that there are two isoforms of CERK with overlapping activity, only one form of which is knocked out in mutated mice. Certainly, sphingosine-1-phosphate is not N-acylated in mammalian cells, nor does there appear to be an enzyme equivalent to phospholipase D (i.e. a 'sphingomyelinase D').
Catabolism: The reverse reaction to produce ceramide is accomplished by phosphatases, suggesting that ceramide and ceramide-1-phosphate are readily interconvertible in cells. The enzymes that have been implicated include a specific ceramide-1-phosphate phosphatase, phosphatidate phosphohydrolase, and the lysosomal acid sphingomyelinase.
It is now known that ceramide-1-phosphate possesses a number of biological functions, some of which are confined to specific cell types and are very different from those of other sphingolipid metabolites. In contrast to sphingosine-1-phosphate it is not secreted by intact cells, although it is released by leaky or damaged cells. Some of these functions may be a result of its physical properties in that it is fusogenic, increasing the fusibility of vesicle membranes. It is not believed to participate in raft formation in membranes. As ceramide-1-phosphate and ceramide have antagonistic functions, as discussed below, a correct balance between the concentrations of the two metabolites is essential for cell and tissue homeostasis. The relative concentrations of sphingosine-1-phosphate and long-chain bases must also be considered, as all are mutually convertible as part of the 'sphingolipid rheostat' (discussed further in relation to ceramides and sphingosine-1-phosphate). A consequence of distortion of this balance in any direction may be metabolic dysfunction or disease, as the activities of the enzymes involved in synthesis and catabolism must be coordinated efficiently to ensure that cells function normally.
Ceramide-1-phosphate is a key regulator of cell growth and survival and stimulates DNA synthesis and cell division in rat fibroblasts. Like sphingosine-1-phosphate, it is a potent inhibitor of apoptosis and promoter of cell survival. For example in macrophages, ceramide-1-phosphate blocks apoptosis through inhibition of the enzyme acid sphingomyelinase, which generates the pro-apoptotic molecule ceramide. It also inhibits serine palmitoyltransferase, the key regulatory enzyme in the biosynthesis of long-chain bases and thence of ceramides. Similarly, by inhibiting caspase activities and preventing DNA fragmentation in macrophages, it prevents apoptosis. Ceramide-1-phosphate is released from damaged cells and is believed to have a role in the recruitment of stem/progenitor cells to damaged organs and may promote their vascularization with the potential to function in regenerative medicine. Ceramides inhibit cell proliferation stimulated by this means by up-regulation of the lipid phosphate phosphatase activity that leads to dephosphorylation of ceramide-1-phosphate.
Similarly, ceramide-1-phosphate is an important mediator of inflammation by stimulating the release of arachidonic acid through activation of the specific cytosolic phospholipase A2α (cPLA2α), which is the initial rate-limiting enzyme in the production of the inflammatory prostaglandins and leukotrienes via the release of arachidonic acid. The discovery of this role of ceramide-1-phosphate arose from the finding that an important component of the venom from the spider Loxosceles reclusus is the enzyme sphingomyelinase D (also present in some bacteria but not mammalian cells), which hydrolyses sphingomyelin to ceramide-1-phosphate, and causes a severe inflammatory response mediated by prostaglandins. Ceramide-1-phosphate activates phospholipase A2 by binding with it directly via a Ca2+-dependent phospholipid binding domain, as opposed to indirectly via a receptor mechanism; the effect is to translocate the enzyme from the cytosolic compartment to the intracellular membranes where phospholipid substrates for eicosanoid production such as phosphatidylcholine are located. There is evidence that ceramide kinase and phospholipase A2 activities are closely linked within the same membranes, mainly the trans-Golgi network, following recruitment of the latter enzyme from the cytosol. As an example, by stimulating the biosynthesis of pro-inflammatory eicosanoids, ceramide-1-phosphate promotes the inflammatory phase of wound repair and inhibits the proliferation and remodelling steps. As the transport protein CPTP transfers ceramide-1-phosphate between membranes, it contributes to the regulation of eicosanoid production. There may be synergy with the activity of sphingosine-1-phosphate, which induces up-regulation of the enzyme cyclooxygenase-2 (COX-2).
Conversely, ceramide-1-phosphate has anti-inflammatory properties when produced in specific cell types or tissues in that it inhibits the release of pro-inflammatory cytokines and blocks activation of the pro-inflammatory transcription factor NF-κB and the formation of tumor necrosis factor α (TNFα), which if produced to excess can be contributors to the deleterious effects of septic shock. It thus has the opposite effect to ceramides. Also, it stimulates the migration of macrophages and increases their release of anti-inflammatory interleukin-10. Inhibition of acid sphingomyelinase and the subsequent reduction of ceramide levels by ceramide-1-phosphate production may be required for the resolution of inflammation and infection in the lung.
There is some evidence that ceramide-1-phosphate binds to and activates a plasma membrane receptor that is different from the receptors for sphingosine-1-phosphate, but this has not been fully characterized. Sphingosine-1-phosphate functions mainly via G-protein-coupled receptors, and this is now believed to be true for certain functions of ceramide-1-phosphate, e.g. macrophage migration. However, the latter lipid also acts by binding directly to its target molecules, for example to phospholipase A2 (see above). A direct interaction with a receptor at the cell surface may not always occur, and although ceramide-1-phosphate added exogenously induces a number of cellular responses in vitro, it is believed that some of these effects are a result of ceramide generated on the plasma membrane via hydrolysis of ceramide-1-phosphate. There are apparently contradictory effects during adipogenesis in that although ceramide kinase is upregulated during differentiation of pre-adipocytes into mature adipocytes, exogenous ceramide-1-phosphate reduces adipogenesis by acting through a putative Gi protein-coupled receptor. CERK may also regulate the biogenesis of lipid droplets. In relation to obesity, experiments with animal models have shown that deletion of CERK suppresses the inflammatory cytokines associated with high-fat diets and returns insulin signalling to normal.
In addition, there is increasing evidence that ceramide-1-phosphate has a negative role in cancer and obesity, and that it has functions in the nervous and immune systems. Both sphingosine-1-phosphate and ceramide-1-phosphate are potent chemo-attractants for a variety of cell types with effects upon the trafficking of normal and malignant cells, but especially of normal hematopoietic stem/progenitor cells. In particular, ceramide-1-phosphate has been shown to be involved in cancer cell growth, migration and survival, although it is not yet known whether it participates in inflammation-associated cancer. CERK is overexpressed in breast cancer and is associated with poor prognosis; it is involved in the migration and invasion of cancer cells in the pancreas and lung. It is hoped that pharmacological control of the activities of this enzyme may inhibit or prevent cancer metastasis.
3. Phytoceramide-1-Phosphate in Plants
Phytoceramide-1-phosphate with 2-hydroxy fatty acids as the N-acyl constituents and phytosphingosine as the sphingoid base is formed in plant tissues, such as Arabidopsis thaliana, where it is generated from ceramide by a ceramide kinase (CERK/ACD5) and also from the important membrane constituents the glycosylinositol phosphoceramides (mainly the species with two sugar residues) by a specific phospholipase D. However, it is produced at such low levels in undamaged tissues that it is not easily measured in vivo. Ceramide kinase is not present in yeast.
It may be important for growth and to remove excess ceramide to make the plant more resistant to environmental stress, especially the response to cold; phosphorylated ceramides accumulate rapidly if transiently upon cold shock treatment, and CERK/ACD5 promotes seed germination at low temperatures. Similarly, the balance between ceramide and phytoceramide-1-phosphate may be crucial in modulating the process of apoptosis in plants as the latter is believed to be pro-survival. A ceramide-1-phosphate phosphatase that might convert ceramide-1-phosphate to the free form has not yet been identified. Arabidopsis mutants that lack ceramide kinase have impaired defense mechanisms and display spontaneous apoptosis. The mechanisms for such signalling effects in plants have still to be determined.
Analysis of the various components of the sphingomyelin cycle, including ceramide-1-phosphate, can now be carried out in a comprehensive manner by high-performance liquid chromatography in conjunction with tandem mass spectrometry and electrospray ionization. However, a popular method in which a strong alkaline treatment is used to cleave interfering glycerolipids must be followed by a neutralization step, otherwise there can be a gross overestimation of ceramide-1-phosphate levels.
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