Blood lymphocyte numbers, essential for the development of efficient immune responses, are maintained by recirculation through secondary lymphoid organs. We show that lymphocyte trafficking is altered by the lysophospholipid sphingosine-1-phosphate (S1P) and by a phosphoryl metabolite of the immunosuppressive agent FTY720. Both species were high-affinity agonists of at least four of the five S1P receptors. These agonists produce lymphopenia in blood and thoracic duct lymph by sequestration of lymphocytes in lymph nodes, but not spleen. S1P receptor agonists induced emptying of lymphoid sinuses by retention of lymphocytes on the abluminal side of sinus-lining endothelium and inhibition of egress into lymph. Inhibition of lymphocyte recirculation by activation of S1P receptors may result in therapeutically useful immunosuppression.

The sphingolipid metabolites ceramide and sphingosine-1-phosphate are second messengers with opposing roles in mammalian cell growth arrest and survival; their relative cellular level has been proposed to be a rheostat that determines the fate of cells. This report demonstrates that this rheostat is an evolutionarily conserved stress-regulatory mechanism that inf luences growth and survival of yeast. Although the role of sphingosine-1-phosphate in yeast was not previously examined, accumulation of ceramide has been shown to induce G 1 arrest and cell death. We now have identified a gene in Saccharomyces cerevisiae, LBP1, that regulates the levels of phosphorylated sphingoid bases and ceramide. LBP1 was cloned from a yeast mutant that accumulated phosphorylated long-chain sphingoid bases and diverted sphingoid base intermediates from sphingolipid pathways to glycerophospholipid biosynthesis. LBP1 and its homolog, LBP2, encode very hydrophobic proteins that contain a novel-conserved sequence motif for lipid phosphatases, and both have long-chain sphingoid base phosphate phosphatase activity. In vitro characterization of Lbp1p shows that this phosphatase is Mg 2؉ -independent with high specificity for phosphorylated long-chain bases, phytosphingosine and sphingosine. The deletion of LBP1 results in the accumulation of phosphorylated long-chain sphingoid bases and reduced ceramide levels. Moreover, deletion of LBP1 and LBP2 results in dramatically enhanced survival upon severe heat shock. Thus, these phosphatases play a previously unappreciated role in regulating ceramide and phosphorylated sphingoid base levels in yeast, and they modulate stress responses through sphingolipid metabolites in a manner that is reminiscent of their effects on mammalian cells.Branching pathways of sphingolipid metabolism may mediate growth arrest, stress, or proliferative responses depending on the cell type and the nature of the stimulus. Ceramide is emerging as an important regulatory component of stress responses and programmed cell death, known as apoptosis (1-5). In contrast, another sphingolipid metabolite, sphingosine-1-phosphate (SPP), has been implicated as a second messenger in cellular proliferation (6) and antagonizes ceramide-mediated apoptosis (7). Thus, it has been suggested that the relative intracellular levels of ceramide and SPP are a critical factor for cell survival. Although the ceramide͞SPP rheostat may be an inherent characteristic of mammalian cells, external stimuli can reset this ratio (7-9). A variety of stress stimuli, including Fas ligand, TNF-␣, IL-1, growth factor withdrawal, anticancer drugs, oxidative stress, heat shock, and ionizing radiation, increase ceramide levels (1, 2, 10, 11), whereas platelet-derived growth factor and other growth factors stimulate rapid, transient elevations in SPP levels (6). The mechanisms that regulate the levels of these sphingolipid second messengers are under intense investigation with most of the attention focused on degradative pathways: sphingomyelinase, which...

Sphingosine and sphingosine-1-phosphate (SPP) are interconvertible sphingolipid metabolites with opposing effects on cell growth and apoptosis. Based on sequence homology with LBP1, a lipid phosphohydrolase that regulates the levels of phosphorylated sphingoid bases in yeast, we report here the cloning, identification, and characterization of a mammalian SPP phosphatase (mSPP1). This hydrophobic enzyme, which contains the type 2 lipid phosphohydrolase conserved sequence motif, shows substrate specificity for SPP. Partially purified Myc-tagged mSPP1 was also highly active at dephosphorylating SPP. When expressed in yeast, mSPP1 can partially substitute for the function of LBP1. Membrane fractions from human embryonic kidney HEK293 cells transfected with mSPP1 markedly degraded SPP but not lysophosphatidic acid, phosphatidic acid, or ceramide-1-phosphate. Enforced expression of mSPP1 in NIH 3T3 fibroblasts not only decreased SPP and enhanced ceramide levels, it also markedly diminished survival and induced the characteristic traits of apoptosis. Collectively, our results suggest that SPP phosphohydrolase may regulate the dynamic balance between sphingolipid metabolite levels in mammalian cells and consequently influence cell fate.S phingosine-1-phosphate (SPP) is a bioactive sphingolipid metabolite that regulates diverse biological processes (reviewed in refs. 1 and 2). Many of its pleiotropic actions appear to be mediated by a family of specific cell surface G proteincoupled receptors (GPCR), known as EDG (endothelial differentiation genes) receptors. Binding of SPP to EDG-1 expressed on endothelial cells enhances survival (3), chemotaxis, and in vitro angiogenesis (4), and adherens junction assembly leading to morphogenetic differentiation (5), whereas binding of SPP to EDG-5 and EDG-3 induces neurite retraction and soma rounding (6, 7). SPP induces activation of G i -gated inward rectifying K ϩ -channels in atrial myocytes (8) and inhibits motility of melanoma cells (9) through as yet uncharacterized GPCRs.SPP also plays important roles inside cells. In response to diverse external stimuli, sphingosine kinase, the enzyme that catalyzes the phosphorylation of sphingosine to SPP, is activated (10-15). Intracellular SPP, in turn, mobilizes calcium from internal stores independently of inositol triphosphate (15, 16), as well as eliciting diverse signaling pathways leading to proliferation (17, 18) and suppression of apoptosis (19)(20)(21)(22).Because of its dual function as a ligand and second messenger and its pivotal role in cell growth and survival, the synthesis and degradation of SPP should be tightly regulated in a spatialtemporal manner. Until recently, however, little was known of the enzymes involved in SPP metabolism. We have purified sphingosine kinase to apparent homogeneity from rat kidney (23) and subsequently cloned and characterized a mammalian sphingosine kinase (24), which belongs to a highly conserved gene family (24,25). Enforced expression of sphingosine kinase markedly enhanced proliferation a...

Potent antifungal activity was detected in fermentation extracts of Sporormiella australis and two related components were isolated from solid fermentations using silica gel and high speed countercurrent chromatography. The most active antifungal component, australifungin, contained a unique combination of a-diketone and jS-ketoaldehyde functional groups. Australifungin exhibited broad spectrum antifungal activity against humanpathogenic fungi with MICs against Candida spp., Cryptococcus neoformans, and Aspergillus spp. between 0.01 5 and 1.0 //g/ml. Modeof action studies revealed that australifungin interfered with fungal lipid metabolism by specifically inhibiting sphingolipid synthesis at the step converting sphinganine to ceramide.