CHAPTER 2
SPHINGOLIPIDS
2.1 SPHINGOLIPIDS
For long time lysophospholipids have been considered as simple costituents of cellular membrane, but recently it a growing evidence shows that they are also bioactive signalling molecules. The most widely studied signalling lysophospholipids are 1,2-diacylglycerol (DAG), arachidonic acid and its metabolites, phosphoinositol 3, 4,5 tri-phosphate (PIP3), and recently also sphingolipids as lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P). These bioactive lipids influence a diverse range of cellular processes, including proliferation, survival, adhesion, migration, morphogenesis and differentiation. As outlined in recent reviews, it has now been established that LPA and S1P play important roles in the immune [68-71], cardiovascular [72-76], nervous [74,77– 79], reproductive [74,80–83], and respiratory [75,82–84], systems, as well as in cancer.[85-89] Here we add further support to the notion that the physiological and pathophysiological actions of lysophospholipids are yet to be fully realised, and highlight their emerging role in the stem cell/progenitor system.
2.2 BIOSYNTHESIS AND METABOLISM OF S1P
Studies of the least 15 years have clearly demonstrated that S1P is generated by sequential action of sphingomyelinases and ceramidase on sphingomyelin and ceramide respectively, followed by sphingosine kinase- (SphK-) directed phosphorylation of sphingosine to S1P [90-92].S1P can either be converted back to sphingosine by specific S1P phosphatases and non-specific LPPs, or degraded by S1P lyase to form phosphoethanolamine and hexadecenal (Fig. 3). So in agreement with the role of S1P as autocrine and paracrine regulator of key biological events, S1P metabolism appears to be strictly regulated.
Two mammalian sphingosine kinases have been identified (SK1 and SK2), each with a variety of alternatively spliced isoforms that differ at their N-termini. SK1 and SK2 originate from different genes, possess a high degree of sequence similarity but differ in protein size, tissue distribution, developmental expression, catalytic properties and somewhat in their substrate specificity [92]. Interestingly, the two sphingosine kinases appear to have distinct biological roles, with SK1 promoting cell growth and survival [93,94] while SK2 enhances apoptosis [95,96]. While the mechanisms responsible for the divergent roles of the two sphingosine kinases have not been definitively established, current evidence suggests that the observed differential subcellular localisation of these enzymes may be a major determinant of their functions [92,97]. Following stimulation of cells with a variety of growth factors and cytokines, SK1 is activated and translocates from the cytoplasm to the plasma membrane where it appears the major source of S1P secreted from cells under these conditions [98,99]. Interestingly, SK1 can also be secreted where it may generate S1P extracellularly [100-102]. Thus, SK1 appears a major contributor to extracellular S1P. In contrast, SK2 is found mainly associated with cellular organelles, including the endoplasmic reticulum and nucleus [95,97], with the exact function of this localisation yet to be clearly elucidated.
S1P breakdown appears also to be quite complex; S1P dephosphorylation can be mediated by two S1P-specific phosphohydrolases, SPP1 and SPP2 [103,104]. Both SPPs are localised to the endoplasmic reticulum, with their catalytic sites predicted to face the luminal site of this organelle [104,105]. Interestingly, at this location the sphingosine formed from SPP action appears to be directed to the biosynthesis of ceramide through ceramide synthase present on the cytoplasmic face of the endoplasmic reticulum [105]. Thus, the SPPs may assist in regulating the balance between S1P and ceramide and the opposing actions of these sphingolipids on apoptosis [105]. S1P can also be degraded by S1P lyase, a pyridoxal-5-phosphate-dependent enzyme that, like the SPPs, is localised to the endoplasmic reticulum [106]. Unlike the SPPs, however, the catalytic site of S1P lyase is located on the cytoplasmic face of this organelle [107]. In this location S1P lyase appears to
not only assist in controlling the levels of intracellular S1P, but is also an important contributor to the regulation of extracellular S1P levels in tissues [108]. So both the catabolic pathways of S1P appear to be regulated and capable of profoundly influencing S1P cellular content and biological activity [109,110].
FIG.3 Pathways of sphingosine-1-phosphate metabolism. Key enzymes for the formation and
degradation of S1P are shown
2.3 S1P SIGNALLING
S1P is abundantly stored in platelets, which have high levels of sphingosine kinase and lack S1P lyase, and is released on platelet activation [111]. Recent studies also implicate red blood cells as a major source of S1P [112,113], while S1P release has also been observed in a diverse range of other cells [111] often in response to cell stimuli that activate SK1, such as growth factors and cytokines [98]. Recent evidence suggests S1P release is mediated by ATP binding cassette (ABC) transporters, and in mast cells, specifically ABCC1 [114]. Together, this results in high levels of S1P in circulation, with S1P levels in human serum and plasma estimated to be 0.4–1.2 and 0.2–0.5 M, respectively [115], with most of this bound to high density lipoproteins and other lipoproteins, or carrier proteins such as serum albumin.[111,115]
S1P have effects on most cells, with the most common cellular responses involving proliferation, cell survival, migration and differentiation. It is presently widely accepted that in mammals S1P exerts its biological effects through ligation to at least five different specific receptors, previously known as EDG (Endothelial Differentiation Gene) receptors and now renamed S1P receptors (S1PRs) [116], whereas in invertebrates no sphingolipid-specific receptors have been so far identified in favour of an exclusively intracellular mechanism of action [91]. These receptors are
differentially expressed and linked to different G proteins, allowing S1P to elicit a variety of somewhat cell-specific responses through the activation of classic Gi, Gq and G12 signalling pathways. These signalling pathways have been covered extensively in a number of excellent recent reviews [69,82,117-122] and therefore will only be briefly discussed here.
The S1P GPCRs display considerable complexity in their signalling functions, (Fig. 4) showing both redundancy and functional antagonism (reviewed in [119]. S1P1 appears to only couple to Gi
proteins to activate a number of signalling pathways, including ERK1/2 to enhance cell proliferation, Akt to enhance survival, and Rac to enhance migration [82,120,122]. S1P2 couples to
Gi, Gq and G12 proteins, and like S1P1 stimulates ERK1/2, but also activates Rho and inhibits Rac,
thus promoting stress fibre formation and inhibiting cell migration [82,120,122]. S1P3 also couples
to Gi, Gq and G12 proteins, but elicits different signalling to S1P2 since it activates ERK1/2, PLC,
Akt, Rho and Rac [82,120,122]. S1P4 couples to Gi and G12 proteins and activates ERK1/2, PLC,
AC, Rho and Rac [123,124]. S1P5 also shows functional antagonism since, unlike the other S1P
GPCRs it inhibits ERK1/2 to decrease cell growth [125].
Intriguingly, the receptor-dependent biological response elicited by S1P in a specific cell type appears to be critically dependent on the typology and the expression levels of receptor subtypes since some of them are recognized to mediate opposite biological effects. Besides its action as ligand of S1PRs, in Mammals S1P, similarly to other bioactive lipids, exerts some of its effects as intracellular mediator in a receptor-independent manner. [90] Noticeably, it is widely accepted that the biological activity of a number of growth factors and cytokines depends at least in part on the specific stimulation of S1P formation. [90] Moreover, to further support the notion that S1P is an important regulator of cell physiology, it has been proved that it largely accounts for the atheroprotective effects exerted by high-density lipoproteins. [126]. In agreement with the fundamental role of cytoskeleton in the regulation of cell shape and motility, S1P has been demonstrated to induce profound cytoskeletal rearrangements in various cell systems, strictly associated to the final biological response elicited by the sphingolipid.
FIG.4
2.4 S1P IN THE REGULATION OF ADULT HEMATOPOIETIC STEM CELLS AND VASCULAR PROGENITORS
Although still emerging, recent evidence suggests sphingolipids play an important role in the regulation of a variety of these adult stem cells and their progenitors.
Two lineages of stem cells form from the hemangioblast, the hematopoietic and vascular stem cells. From the hematopoietic stem cells, two principle cell types are derived, the lymphoid (T, B and NK) and the myeloid (neutrophils, monocytes, erythrocytes, platelets and dendritic cells) lineages. In contrast, the vascular stem cells differentiate to produce both endothelial cells and pericytes (reviewed in [127]. Whetton et al. [129] were the first to catalogue the expression of S1P receptors in murine primitive hematopoietic stem cells. Using the seminal markers for murine progenitor cells (i.e. lineage−/Sca-1+/c-Kit+), they observed that primitive Lin−Sca+Kit+ hematopoietic stem cells expressed S1P1–4, but not S1P5. In contrast, the less primitive Kit− cells expressed all five known
S1P receptors, including S1P5. Thus, the expression of S1P receptors changes during hematopoietic
cell differentiation, although the functional consequences of this are yet to be elucidated. S1P induced migration of the Kit+ and Kit− hematopoietic stem cells in vitro, although the effect was greater on the generally less mobile primitive Kit−cells. Furthermore, S1P substantially enhanced the chemotactic migratory response of the primitive hematopoietic stem cells to stromal-derived factor 1 (SDF-1) through a mechanism involving the activation of the Rac, Rho and Cdc42 G
proteins, that is controlled in part by PI3K and the Rac/Rho/Cdc42 guanyl nucleotide exchange factor vav-1. [129]. This data suggest that S1P provide a stimulus for these primitive hematopoietic stem cells to become more motile, leave the hematopoietic stem cell niche microenvironment and relocate to fresh sites where hematopoiesis can occur.
The effects of S1P on human hematopoietic progenitor cells are also known. Kimura et al. [130] compared S1P receptor expression in primitive CD34+CD38− hematopoietic progenitors and the more committed CD34+CD38+ cells. While S1P1 was expressed in both subsets, S1P3 and S1P5
were expressed only in CD34+CD38− progenitors, while S1P2 and S1P4 were found only in
CD34+CD38+ cells. Again, however, the functional consequences of the differential expression of the S1P receptors in these cells remains to be elucidated. Activation of S1P receptors by FTY720 augments SDF-1-dependent transendothelial progenitor cell migration in vitro and bone marrow homing. [129]. In support of these findings, Seitz et al. [131] demonstrated that S1P can act as a direct chemoattractant for human CD34+ progenitors in vitro. Furthermore, sustained S1P1
activation in vivo, via FTY720, increased engraftment of human CD34+ progenitor cells in NOD/SCID mice. [129] Similarly, Walter et al. [132] recently demonstrated that human endothelial progenitor cells treated with S1P, or with FTY720, increased flow in ischemic murine limbs via the S1P3 receptor. This was supported by studies showing a decrease in blood flow following an
ischemic event in mice lacking the S1P3 receptor [131]. Together, these studies provide an
emerging picture for the importance of S1P in regulating the mobilization and homing of hematopoietic stem cells and progenitors.
