dihydrosphingosine-1-phosphate and phytosphingosine

Sphingolipids have been suggested to act as second messengers for an array of cellular signaling activities in plant cells, including stress responses and programmed cell death (PCD). However, the mechanisms underpinning these processes are not well understood. Here, we report that an Arabidopsis mutant, fumonisin B1 resistant 11-1 (fbr 11-1), which fails to generate reactive oxygen intermediates (ROIs), is incapable of initiating PCD when the mutant is challenged by fumonisin B(1) (FB(1)), a specific inhibitor of ceramide synthase. Molecular analysis indicated that FBR11 encodes a long-chain base 1 (LCB1) subunit of serine palmitoyltransferase (SPT), which catalyzes the first rate-limiting step of de novo sphingolipid synthesis. Mass spectrometric analysis of the sphingolipid concentrations revealed that whereas the fbr 11-1 mutation did not affect basal levels of sphingoid bases, the mutant showed attenuated formation of sphingoid bases in response to FB(1). By a direct feeding experiment, we show that the free sphingoid bases dihydrosphingosine, phytosphingosine and sphingosine efficiently induce ROI generation followed by cell death. Conversely, ROI generation and cell death induced by dihydrosphingosine were specifically blocked by its phosphorylated form dihydrosphingosine-1-phosphate in a dose-dependent manner, suggesting that the maintenance of homeostasis between a free sphingoid base and its phosphorylated derivative is critical to determining the cell fate. Because alterations of the sphingolipid level occur prior to the ROI production, we propose that the free sphingoid bases are involved in the control of PCD in Arabidopsis, presumably through the regulation of the ROI level upon receiving different developmental or environmental cues.

Topics: Apoptosis; Arabidopsis; Arabidopsis Proteins; Fumonisins; Genome, Plant; Mutation; Phosphorylation; Protein Subunits; Reactive Oxygen Species; Serine C-Palmitoyltransferase; Signal Transduction; Sphingolipids; Sphingosine

Sphingolipids participate in membrane structure and signaling in neuronal cells, and an emerging strategy for control of gliomas is to inhibit growth and/or induce apoptosis using ceramide and ceramide analogs. Nonetheless, some sphingolipids (ceramides and sphingosine) induce and others (sphingosine 1-phosphate) inhibit apoptosis; therefore, when testing putative anti-cancer agents, it is critical to obtain precise knowledge of the types and quantities of not only the test compounds, but also their effects on endogenous species. Combination of liquid chromatography and tandem mass spectrometry affords a "metabolomic" profile of all of the intermediates of ceramide biosynthesis (3-ketosphinganine, sphinganine and dihydroceramides) and the direct products of ceramide metabolism (sphingomyelins and monohexosylceramides as well as sphingosine and sphingosine 1-phosphate). This method has been applied to four human glioma cell lines (LN18, LN229, LN319 and T98G), and differences in the amounts and types of sphingolipids were found. For example, LN229 and LN319 have approximately twice the sphingosine 1-phosphate of LN18 and T98G; LN229 and LN319 have more monohexosylceramides than lactosylceramides, whereas the opposite is the case for LN18 and T98G; and the fatty acyl chain distributions of the sphingolipids differ among the cell lines. The ability to obtain this type of "metabolomic" profile allows studies of how anti-cancer agents (especially sphingolipids and sphingolipid analogs) affect the amounts of these bioactive species, and may lead to a better understanding of the abnormal phenotypes of gliomas.

Topics: Astrocytoma; Cell Line, Tumor; Ceramides; Chromatography, High Pressure Liquid; Fatty Acids; Galactosylceramides; Glioblastoma; Glioma; Glucosylceramides; Humans; Lactosylceramides; Lysophospholipids; Molecular Structure; Spectrometry, Mass, Electrospray Ionization; Sphingolipids; Sphingomyelins; Sphingosine

Topics: Acetyltransferases; Acyltransferases; Chromatography, Thin Layer; Fungal Proteins; Growth Inhibitors; Membrane Proteins; Mixed Function Oxygenases; Phosphoric Monoester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Serine C-Palmitoyltransferase; Signal Transduction; Sphingolipids; Sphingosine