Participation of de novo sphingolipid biosynthesis in the regulation of autophagy in response to diverse agents

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Sims, Kacee Hall
Merrill, Alfred H.
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Sphingolipids are a complex family of molecules that participate in many aspects of cell structure and function, including an essential cellular process known as autophagy. Autophagy is a degradation and recycling pathway whereby intracellular components are sequestered into double-membrane vesicles, known as autophagosomes, for subsequent fusion with lysosomes and degradation. Autophagy takes part in cell survival, host immune defense against pathogens, and other biological processes, but is also sometimes lethal. Ceramide, sphingosine 1-phosphate, and more recently dihydroceramide have been shown to induce autophagy, which opens an interesting new field of cell regulation by sphingolipids. This dissertation describes two new cases in which sphingolipids participate in the induction of autophagy: a) RAW264.7 cells treated with Kdo2-Lipid A, a lipopolysaccharide sub-structure with endotoxin activity equal to LPS; and b) MCF7 cells treated with fenretinde, a chemotherapeutic agent which has shown success in clinical trials. It also analyzes the structural properties of fenretinide that contribute to its ability to modulate sphingolipid metabolism through inhibition of dihydroceramide desaturase, thereby elevating dihydroceramide and induction of autophagy. Autophagy was monitored by following the redistribution of GFP-LC3 into discrete punctate vesicles in response to the agents and by Western blotting; in parallel, the sphingolipid composition of the cells was monitored by liquid chromatography, electrospray ionization tandem mass spectrometry. These analyses revealed that Kdo2-Lipid A and fenretinide induce profound changes in sphingolipid metabolism in RAW264.7 and MCF7 cells, respectively, and that one of the purposes for increased de novo biosynthesis is to enable the production of autophagosomes, as the autophagic response was inhibited by myriocin. These studies have uncovered a direct link between sphingolipid metabolism and autophagy, which could pave the way for new therapeutic interventions for the treatment of pathogenic infection and be clinically useful in enhancing the efficacy of current cancer treatment strategies.
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