Sphingosine-1-phosphate (S1P), a lipid signaling molecule that regulates many cellular functions, is synthesized from sphingosine and ATP by the action of sphingosine kinase. Two such kinases have been identified, SPHK1 and SPHK2. To begin to investigate the physiological functions of sphingosine kinase and S1P signaling, we generated mice deficient in SPHK1. Sphk1 null mice were viable, fertile, and without any obvious abnormalities. Total SPHK activity in most Sphk1؊/؊ tissues was substantially, but not completely, reduced indicating the presence of multiple sphingosine kinases. S1P levels in most tissues from the Sphk1؊/؊ mice were not markedly decreased. In serum, however, there was a significant decrease in the S1P level. Although S1P signaling regulates lymphocyte trafficking, lymphocyte distribution was unaffected in lymphoid organs of Sphk1؊/؊ mice. The immunosuppressant FTY720 was phosphorylated and elicited lymphopenia in the Sphk1 null mice showing that SPHK1 is not required for the functional activation of this sphingosine analogue prodrug. The results with these Sphk1 null mice reveal that some key physiologic processes that require S1P receptor signaling, such as vascular development and proper lymphocyte distribution, can occur in the absence of SPHK1.Sphingosine-1-phosphate (S1P) 1 is a signaling molecule that influences cellular functions including proliferation, survival, migration, adhesion molecule expression, and morphogenesis (1-4). S1P binds to members of the S1P receptor family (also known as EDG receptors) and, via G proteins, triggers multiple signaling pathways (5, 6). S1P has also been shown to function intracellularly mediating calcium homeostasis, cell growth, and suppression of apoptosis (7,8). In mammals, vascular development and lymphocyte trafficking are dependent on S1P receptor signaling (9 -13).Sphingosine kinase (SPHK) catalyzes the synthesis of S1P via the phosphorylation of sphingosine. SPHK activity is elevated by several stimuli, including platelet-derived growth factor, vascular endothelial growth factor, tumor necrosis factor-␣, and phorbol ester, which trigger an increase in cellular S1P levels (14). Sphk genes have been identified in mammals (15-18), insects (19), plants (20), yeast (21), worm (22), and slime mold (23, 24). Mammals carry two known SphK genes, which in mice are encoded by Sphk1 and Sphk2. The two enzymes contain five highly conserved regions (C1-C5) and an ATP binding site within a conserved lipid kinase catalytic domain (15, 16). SPHK1 has a predominantly cytoplasm localization but can be induced to localize to the inner leaflet of the plasma membrane. Interestingly in endothelial cells SPHK1 is secreted and is capable of producing S1P extracellularly (25). Sphk1 shows a tissue distribution and developmental expression pattern different from Sphk2, although both enzymes are widely expressed (16,26).The importance of S1P receptor signaling in lymphocyte trafficking was first illuminated by the activities of FTY720, a potent immunosuppressive agent. FT...

Diverse insect species harbor symbiotic bacteria, which play important roles such as provisioning nutrients and providing defense against natural enemies [1-6]. Whereas nutritional symbioses are often indispensable for both partners, defensive symbioses tend to be of a facultative nature [1-12]. The Asian citrus psyllid Diaphorina citri is a notorious agricultural pest that transmits Liberibacter spp. (Alphaproteobacteria), causing the devastating citrus greening disease or Huanglongbing [13, 14]. In a symbiotic organ called the bacteriome, D. citri harbors two distinct intracellular symbionts: a putative nutrition provider, Carsonella_DC (Gammaproteobacteria), and an unnamed betaproteobacterium with unknown function [15], for which we propose the name "Candidatus Profftella armatura." Here we report that Profftella is a defensive symbiont presumably of an obligate nature with an extremely streamlined genome. The genomes of Profftella and Carsonella_DC were drastically reduced to 464,857 bp and 174,014 bp, respectively, suggesting their ancient and mutually indispensible association with the host. Strikingly, 15% of the small Profftella genome encoded horizontally acquired genes for synthesizing a novel polyketide toxin. The toxin was extracted, pharmacologically and structurally characterized, and designated diaphorin. The presence of Profftella and its diaphorin-biosynthetic genes was perfectly conserved in the world's D. citri populations.

Ceramide (N-acylsphingosine) biosynthesis has been proposed to involve introduction of the 4,5-trans-double bond of sphingosine after synthesis of dihydroceramide (i.e. N-acylsphinganine). For the first time, the in vitro conversion of dihydroceramide to ceramide has been demonstrated using rat liver microsomes and N-[1-14 C]octanoyl-D-erythro-sphinganine (st-H 2 Cer) and either NADH or NADPH as co-substrate; the apparent K m values for st-H 2 Cer and NADH were 340 and 120 M, respectively. Molecular oxygen is required for enzymatic activity, and cyanide, divalent copper, as well as antibodies raised against cytochrome b 5 are inhibitory, which suggests that this enzyme should be named dihydroceramide desaturase based on these similarities with the mechanism of ⌬ 9 -desaturase (stearoyl-CoA desaturase). Factors that influenced the activity of dihydroceramide desaturase include the alkyl chain length of the sphingoid base (in the order C 18 > C 12 > C 8 ) and fatty acid (C 8 > C 18 ); the stereochemistry of the sphingoid base (D-erythro-> L-threo-dihydroceramides); the nature of the headgroup, with the highest activity with dihydroceramide, but some (ϳ20%) activity with dihydrosphingomyelin (activity was not detected with dihydroglucosylceramide, however); and the ability to utilize alternative reductants (ascorbic acid could substitute for a reduced pyridine nucleotide, but was inhibitory at higher concentrations). Dihydroceramide desaturase was inhibited by dithiothreitol, which suggests that it might be possible to alter ceramide synthesis by varying the thiol status of hepatocytes. Consistent with this hypothesis, when rat hepatocytes were cultured in varying concentrations of N-acetylcysteine (5 and 10 mM), there was a decrease in the relative incorporation of [ 14 C]serine into [ 14 C]ceramide. These studies have conclusively established the pathway of ceramide synthesis via desaturation of dihydroceramide and have uncovered several properties of this reaction that warrant further consideration for their relevance to both sphingolipid metabolism and signaling.Over the last few years, it has become clear that ceramide plays important roles in the metabolism of cells. It is involved as a second messenger in what has become known as the sphingomyelin cycle (reviewed in Ref. 1) and, to name but a few, serves as a mediator of cellular senescence (2), apoptosis, and differentiation in many cell types (3). It was also observed that not only ceramide derived from the sphingomyelin pool found in the plasma membrane but also that from de novo synthesis is involved in the cellular responses to inducers of apoptosis (4) and differentiation (5). These results suggest an important role of the anabolic pathway of ceramides in signal transduction. It is therefore very likely that sphingolipid biosynthesis is tightly regulated. Taking into consideration that dihydroceramide does not mimic the effects of ceramide (6, 7), one possible site of regulation might be dihydroceramide desaturase. In addition, ceramide is also involve...

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Cerebellar granule cells from sphingosine 1-phosphate (S1P) lyase-deficient mice were used to study the toxicity of this potent sphingolipid metabolite in terminally differentiated postmitotic neurons. Based on earlier findings with the lyase-stable, semi-synthetic, cis-4-methylsphingosine phosphate, we hypothesized that accumulation of S1P above a certain threshold induces neuronal apoptosis. The present studies confirmed this conclusion and further revealed that for S1P to induce apoptosis in lyase-deficient neurons it must also be produced by sphingosine-kinase2 (SK2). These conclusions are based on the finding that incubation of lyase-deficient neurons with either sphingosine or S1P results in a similar elevation in cellular S1P; however, only S1P addition to the culture medium induces apoptosis. This was not due to S1P acting on the S1P receptor but to hydrolysis of S1P to sphingosine that was phosphorylated by the cells, as described before for cis-4-methylsphingosine. Although the cells produced S1P from both exogenously added sphingosine as well as sphingosine derived from exogenous S1P, the S1P from these two sources were not equivalent, because the former was primarily produced by SK1, whereas the latter was mainly formed by SK2 (as also was cis-4-methylsphingosine phosphate), based on studies in neurons lacking SK1 or SK2 activity. Thus, these investigations show that, due to the existence of at least two functionally distinct intracellular origins for S1P, exogenous S1P can be neurotoxic. In this model, S1P accumulated due to a defective lyase, however, this cause of toxicity might also be important in other cases, as illustrated by the neurotoxicity of cis-4-methylsphingosine phosphate.Sphingosine 1-phosphate (S1P) 2 is a potent lipid mediator that has been shown to regulate a wide range of physiological processes, including proliferation, differentiation, motility, cytoskeleton rearrangements, and calcium homeostasis (1, 2). There is convincing experimental evidence that this bioactive sphingolipid can act both extracellularly, as a ligand for a family of five specific G protein-coupled receptors, and inside the cells, as a second messenger (3, 4). In most cell types described so far, S1P and its metabolic precursor ceramide exert antagonistic effects on cell survival with S1P being generally regarded as a survival signal, whereas ceramide and sphingosine are generally toxic (5, 6). Interestingly, generation of sphingosine and S1P is generally thought to be dependent on the availability of ceramide (7), however, relatively high amounts of S1P are also present in blood, lymph, and cerebrospinal fluid (8, 9) and may serve as additional sources for some cells.More than a decade ago, we introduced the synthetic sphingosine analog cis-4-methylsphingosine as a tool for studies of sphingoid base metabolism and function (10). When added to the culture medium, this analog is taken up and mainly phosphorylated to the respective cis-4-methylsphingosine phosphate, which accumulates intracellularly, because it ...