Sphingolipid metabolites such as sphingosine-1-phosphate (S1P) and ceramide modulate apoptosis during development and in response to stress. In general, ceramide promotes apoptosis, whereas S1P stimulates cell proliferation and protects against apoptosis. S1P is irreversibly degraded by the enzyme S1P lyase (SPL). In this study, we show a crucial role for SPL in mediating cellular responses to stress. SPL expression in HEK293 cells potentiated apoptosis in response to stressful stimuli including DNA damage. This effect seemed to be independent of ceramide generation but required SPL enzymatic activity and the actions of p38 MAP kinase, p53, p53-inducible death domain protein (PIDD), and caspase-2 as shown by molecular and chemical inhibition of each of these targets. Further, SPL expression led to constitutive activation of p38. Endogenous SPL expression was induced by DNA damage in WT cells, whereas SPL knockdown diminished apoptotic responses. Importantly, SPL expression was significantly downregulated in human colon cancer tissues in comparison with normal adjacent tissues, as determined by quantitative real-time PCR (Q-PCR) and immunohistochemical analysis. Down-regulation of S1P phosphatases was also observed, suggesting that colon cancer cells manifest a block in S1P catabolism. In addition, SPL expression and activity were down-regulated in adenomatous lesions of the Min mouse model of intestinal tumorigenesis. Taken together, these results indicate that endogenous SPL may play a physiological role in stress-induced apoptosis and provide an example of altered SPL expression in a human tumor. Our findings suggest that genetic or epigenetic changes affecting intestinal S1P metabolism may correlate with and potentially contribute to carcinogenesis.intestinal tumorigenesis ͉ Min mouse ͉ sphingolipid ͉ etoposide S phingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite and the final common product of complex sphingolipid metabolism. S1P acts through its cognate G proteincoupled receptors to inhibit apoptosis, regulate lymphocyte trafficking and to promote DNA synthesis, cell proliferation, cell migration, and angiogenesis (1, 2). The SPHK1 gene, which encodes the major sphingosine kinase responsible for S1P synthesis, can act as an oncogene in model systems (3). Further, parenteral administration of S1P-specific antibodies markedly slows human cancer xenograft progression and angiogenesis (4). S1P signaling has been implicated in the development of the drug resistant phenotype in cancer cells (5). Together, these findings strongly support a role for S1P signaling in promoting tumorigenesis and cancer progression. Despite these observations, evidence of genetic changes in human cancer tissues that would directly implicate S1P signaling in these processes is lacking. S1P is irreversibly degraded by the pyridoxal 5Ј-phosphatedependent enzyme, S1P lyase (SPL). SPL is highly conserved throughout evolution, is required for maintenance of physiological levels of S1P and other sphingolipid intermediates an...

Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease cases. A mutation in 1 of over 40 monogenic genes can be detected in approximately 30% of individuals with SRNS whose symptoms manifest before 25 years of age. However, in many patients, the genetic etiology remains unknown. Here, we have performed whole exome sequencing to identify recessive causes of SRNS. In 7 families with SRNS and facultative ichthyosis, adrenal insufficiency, immunodeficiency, and neurological defects, we identified 9 different recessive mutations in SGPL1, which encodes sphingosine-1-phosphate (S1P) lyase. All mutations resulted in reduced or absent SGPL1 protein and/or enzyme activity. Overexpression of cDNA representing SGPL1 mutations resulted in subcellular mislocalization of SGPL1. Furthermore, expression of WT human SGPL1 rescued growth of SGPL1-deficient dpl1Δ yeast strains, whereas expression of diseaseassociated variants did not. Immunofluorescence revealed SGPL1 expression in mouse podocytes and mesangial cells. Knockdown of Sgpl1 in rat mesangial cells inhibited cell migration, which was partially rescued by VPC23109, an S1P receptor antagonist. In Drosophila, Sply mutants, which lack SGPL1, displayed a phenotype reminiscent of nephrotic syndrome in nephrocytes. WT Sply, but not the disease-associated variants, rescued this phenotype. Together, these results indicate that SGPL1 mutations cause a syndromic form of SRNS.

Sphingosine-1-phosphate lyase is a widely expressed enzyme that catalyzes the essentially irreversible cleavage of the signaling molecule sphingosine 1-phosphate. To investigate whether sphingosine-1-phosphate lyase influences mammalian cell fate decisions, a recombinant human sphingosine-1-phosphate lyase fused to green fluorescent protein was expressed in HEK293 cells. The recombinant enzyme was active, localized to the endoplasmic reticulum, and reduced baseline sphingosine and sphingosine 1-phosphate levels. Stable overexpression led to diminished viability under stress, which was attributed to an increase in apoptosis and was reversible in a dose-dependent manner by exogenous sphingosine 1-phosphate. In contrast to sphingosine 1-phosphate, the products of the lyase reaction had no effect on apoptosis. Lyase enzymatic activity was required to potentiate apoptosis, because cells expressing a catalytically inactive enzyme behaved like controls. Stress increased the amounts of long-and very long-chain ceramides in HEK293 cells, and this was enhanced in cells overexpressing wild type but not catalytically inactive lyase. The ceramide increases appeared to be required for apoptosis, because inhibition of ceramide synthase with fumonisin B 1 decreased apoptosis in lyase-overexpressing cells. Thus, sphingosine-1-phosphate lyase overexpression in HEK293 cells decreases sphingosine and sphingosine 1-phosphate amounts but elevates stress-induced ceramide generation and apoptosis. This identifies sphingosine-1-phosphate lyase as a dual modulator of sphingosine 1-phosphate and ceramide metabolism as well as a regulator of cell fate decisions and, hence, a potential target for diseases with an imbalance in these biomodulators, such as cancer.Sphingosine 1-phosphate (S1P) 1 is a sphingolipid metabolite that regulates cell migration, survival, differentiation, angiogenesis, and development. S1P has been shown to activate an extracellular signaling pathway mediated through a family of specific G protein-coupled receptors (1, 2). However, evidence indicates that S1P also mediates effects through a receptorindependent mechanism by functioning as a second messenger within cells (3). Intracellular S1P levels are regulated primarily by three highly conserved enzymes: sphingosine kinase (SPHK), which catalyzes the phosphorylation of sphingosineproducing S1P, S1P phosphatase (S1PP), which reverses the former reaction, and S1P lyase (SPL), a PLP-dependent enzyme that catalyzes the essentially irreversible cleavage of S1P at the C 2-3 carbon-carbon bond, yielding ethanolamine phosphate and a long-chain aldehyde (4 -6).One approach to uncovering roles of S1P signaling in biology has been to manipulate expression of the recently cloned genes of S1P metabolism. Toward that end, altered SPL expression in a variety of cell lines and mutant model systems has yielded pronounced effects and severe phenotypes. For example, Saccharomyces cerevisiae dpl1 null mutants, which lack SPL activity, are highly resistant to heat stress and nutrient de...

Sphingosine-1-phosphate (S1P) activates a widely expressed family of G protein-coupled receptors, serves as a muscle trophic factor and activates muscle stem cells called satellite cells (SCs) through unknown mechanisms. Here we show that muscle injury induces dynamic changes in S1P signaling and metabolism in vivo. These changes include early and profound induction of the gene encoding the S1P biosynthetic enzyme SphK1, followed by induction of the catabolic enzyme sphingosine phosphate lyase (SPL) 3 days later. These changes correlate with a transient increase in circulating S1P levels after muscle injury. We show a specific requirement for SphK1 to support efficient muscle regeneration and SC proliferation and differentiation. Mdx mice, which serve as a model for muscular dystrophy (MD), were found to be S1P-deficient and exhibited muscle SPL upregulation, suggesting that S1P catabolism is enhanced in dystrophic muscle. Pharmacological SPL inhibition increased muscle S1P levels, improved mdx muscle regeneration and enhanced SC proliferation via S1P receptor 2 (S1PR2)-dependent inhibition of Rac1, thereby activating Signal Transducer and Activator of Transcription 3 (STAT3), a central player in inflammatory signaling. STAT3 activation resulted in p21 and p27 downregulation in a S1PR2-dependent fashion in myoblasts. Our findings suggest that S1P promotes SC progression through the cell cycle by repression of cell cycle inhibitors via S1PR2/STAT3-dependent signaling and that SPL inhibition may provide a therapeutic strategy for MD.

FTY720 is a novel immunomodulatory agent that inhibits lymphocyte trafficking and prevents allograft rejection. FTY720 is phosphorylated in vivo, and the phosphorylated drug acts as agonist for a family of G protein-coupled receptors that recognize sphingosine 1-phosphate. Evidence suggests that FTY720-phosphate-induced activation of S1P 1 is responsible for its mechanism of action. FTY720 was rationally designed by modification of myriocin, a naturally occurring sphingoid base analog that causes immunosuppression by interrupting sphingolipid metabolism. In this study, we examined interactions between FTY720, FTY720-phosphate, and sphingosine-1-phosphate lyase, the enzyme responsible for irreversible sphingosine 1-phosphate degradation. FTY720-phosphate was stable in the presence of active sphingosine-1-phosphate lyase, demonstrating that the lyase does not contribute to FTY720 catabolism. Conversely, FTY720 inhibited sphingosine-1-phosphate lyase activity in vitro. Treatment of mice with FTY720 inhibited tissue sphingosine-1-phosphate lyase activity within 12 h, whereas lyase gene and protein expression were not significantly affected. Tissue sphingosine 1-phosphate levels remained stable or increased throughout treatment. These studies raise the possibility that disruption of sphingosine 1-phosphate metabolism may account for some effects of FTY720 on immune function and that sphingosine-1-phosphate lyase may be a potential target for immunomodulatory therapy.FTY720 is a novel immunosuppressive agent that modulates lymphocyte trafficking and prevents allograft rejection (1, 2). FTY720 treatment stimulates lymphocyte homing to peripheral lymph nodes and Peyer's patches, resulting in prolonged survival of allograft tissues and both prevention and treatment of various autoimmune diseases in animal models (3). FTY720 induces numerous effects on the immune system including inhibited egress of both naïve and activated CD4 ϩ , CD8 ϩ , and B lymphocytes from peripheral lymphoid organs and thymus, cysteinyl leukotriene-dependent T cell chemotaxis to lymph nodes, peripheral blood lymphopenia, egress of lymphocytes from the spleen, displacement of B cells from the marginal zone of the spleen, decreased ␤1 integrin expression on marginal zone B cells, homing of hematopoietic progenitor cells to the bone marrow, and decreased vascular permeability (4 -12). Importantly, while FTY720 prevents the migration of lymphocytes to allogeneic graft tissue and other sites of inflammation, it does not diminish the activation, proliferation, or effector functions of B and T lymphocytes in response to antigen stimulation (13). Toxicities associated with FTY720 are limited and distinct from those of other immunosuppressive drugs, which makes FTY720 an ideal candidate for combination immunosuppressive transplantation regimens.FTY720 was rationally designed based on chemical modifications of myriocin, a naturally occurring sphingoid base analog that causes immunosuppression by interrupting sphingolipid metabolism (14). FTY720 is phosphoryl...