Sphingosine Phosphate Lyase Expression Is Essential for Normal Development in Caenorhabditis elegans

Sphingolipids are ubiquitous membrane constituents whose metabolites function as signaling molecules in eukaryotic cells. Sphingosine 1-phosphate, a key sphingolipid second messenger, regulates proliferation, motility, invasiveness, and programmed cell death. These effects of sphingosine 1-phosphate and similar phosphorylated sphingoid bases have been observed in organisms as diverse as yeast and humans. Intracellular levels of sphingosine 1-phosphate are tightly regulated by the actions of sphingosine kinase, which is responsible for its synthesis and sphingosine-1-phosphate phosphatase and sphingosine phosphate lyase, the two enzymes responsible for its catabolism. In this study, we describe the cloning of the Caenorhabditis elegans sphingosine phosphate lyase gene along with its functional expression in Saccharomyces cerevisiae. Promoter analysis indicates tissue-specific and developmental regulation of sphingosine phosphate lyase gene expression. Inhibition of C. elegans sphingosine phosphate lyase expression by RNA interference causes accumulation of phosphorylated and unphosphorylated long-chain bases and leads to poor feeding, delayed growth, reproductive abnormalities, and intestinal damage similar to the effects seen with exposure to Bacillus thuringiensis toxin. Our results show that sphingosine phosphate lyase is an essential gene in C. elegans and suggest that the sphingolipid degradative pathway plays a conserved role in regulating animal development

Regulation of Sphingosine-1-phosphate Lyase Gene Expression by Members of the GATA Family of Transcription Factors

Sphingosine-1-phosphate is a bioactive sphingolipid that regulates proliferation, differentiation, migration, and apoptosis. Sphingosine-1-phosphate is irreversibly degraded by the highly conserved enzyme sphingosine-1-phosphate lyase. Recent studies have suggested that sphingosine-1-phosphate lyase expression affects animal development and cell fate decisions. Despite its crucial role, mechanisms affecting expression of sphingosine-1-phosphate lyase remain poorly understood. In this study, regulation of sphingosine-1-phosphate lyase gene expression was investigated in Caenorhabditis elegans, where lyase expression is spatially restricted to cells of the developing and adult gut and is essential for normal development. Deletion analysis and generation of transgenic worms combined with fluorescence microscopy identified a 350-nucleotide sequence upstream of the ATG start site necessary for maximal lyase expression in adult worms. Site-specific mutagenesis of a GATA transcription factor-binding motif in the promoter led to loss of reporter expression. Knockdown of the gut-specific GATA transcription factor ELT-2 by RNA interference similarly led to loss of reporter expression. ELT-2 interacted with the GATA factor-binding motif in vitro and was also capable of driving expression of a Caenorhabditis elegans lyase promoter-{beta}-galactosidase reporter in a heterologous yeast system. These studies demonstrate that ELT-2 regulates sphingosine-1-phosphate lyase expression in vivo. Additionally, we demonstrate that the human sphingosine-1-phosphate lyase gene is regulated by a GATA transcription factor. Overexpression of GATA-4 led to both an increase in activity of a reporter gene as well as an increase in endogenous sphingosine-1-phosphate lyase protein

A Suppressor/Enhancer Screen in Drosophila Reveals a Role for Wnt-Mediated Lipid Metabolism in Primordial Germ Cell Migration

Wnt proteins comprise a large family of secreted ligands implicated in a wide variety of biological roles. WntD has previously been shown to inhibit the nuclear accumulation of Dorsal/NF-κB protein during embryonic dorsal/ventral patterning and the adult innate immune response, independent of the well-studied Armadillo/β-catenin pathway. In this paper, we present a novel phenotype for WntD mutant embryos, suggesting that this gene is involved in migration of primordial germ cells (PGC) to the embryonic gonad. Additionally, we describe a genetic suppressor/enhancer screen aimed at identifying genes required for WntD signal transduction, based on the previous observation that maternal overexpression of WntD results in lethally dorsalized embryos. Using an algorithm to narrow down our hits from the screen, we found two novel WntD signaling components: Fz4, a member of the Frizzled family, and the Drosophila Ceramide Kinase homolog, Dcerk. We show here that Dcerk and Dmulk (Drosophila Multi-substrate lipid kinase) redundantly mediate PGC migration. Our data are consistent with a model in which the activity of lipid phosphate phosphatases shapes a concentration gradient of ceramide-1-phosphate (C1P), the product of Dcerk, allowing proper PGC migration

Sphingosine-1-Phosphate Enhances Satellite Cell Activation in Dystrophic Muscles through a S1PR2/STAT3 Signaling Pathway

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