Ceramide Synthesis Correlates with the Posttranscriptional Regulation of the Sterol-Regulatory Element-Binding Protein

Abstract

Objective-Sterol-regulatory element-binding proteins (SREBPs) regulate transcription of genes of lipid metabolism.Ceramide decreases transcriptionally active SREBP levels independently of intracellular cholesterol levels. Mechanisms of the ceramide-mediated decrease of SREBP levels were investigated. T he sterol-regulatory element-binding proteins (SREBPs) are important transcription factors of genes of lipid metabolism. In sterol depletion, precursor SREBP is translocated from the endoplasmic reticulum by vesicular trafficking to the Golgi apparatus, 1,2 where the transcriptionally active mature SREBP is generated. Mature SREBP binds to sterolregulatory elements (SRE), cis-acting elements in the promoters of genes of cholesterol and fatty acid synthesis. 3 Cholesterol and unsaturated fatty acids are known regulators of transcriptional and posttranscriptional processing of SREBP. We recently reported that unsaturated fatty acidsmediated decreases in SRE-mediated gene transcription are linked to cellular sphingolipid metabolism. 4 Ceramide, a metabolite of sphingomyelin hydrolysis, also regulates mature SREBP levels. Regulation occurs even in the presence of inhibitors of intracellular cholesterol trafficking, suggesting a cholesterol-independent effect. 4 Ceramide has multiple roles ranging from lipid second messenger to the induction of apoptosis, cell growth, and differentiation. 5,6 Cellular ceramide levels are generated either by de novo synthesis from serine and palmitoyl-CoA or through a recycling pathway of sphingolipid hydrolysis. Methods and Results-ExperimentsCeramide also has a role in intracellular protein trafficking, can inhibit coated vesicle formation and exocytosis in Chinese hamster ovary (CHO) cells, 7 and can modulate endocytosis in mammalian cells. 8 In yeast, ongoing ceramide synthesis is critical in the vesicular ER-to-Golgi transport of GPI-anchored proteins. 9 -11 We investigated the effect of ceramide synthesis on SREBP levels and SRE-mediated gene transcription. Because increased ceramide levels decrease mature SREBP protein levels and SRE-mediated gene transcription, 4 it was anticipated that inhibition of ceramide synthesis should increase mature SREBP levels and SRE-mediated gene transcription. Contrary to this hypothesis, inhibition of ceramide synthesis decreases SRE-mediated gene transcription. Increasing ceramide synthesis correlates with increased mature SREBP levels and SRE-mediated gene transcription. Consequently, the effect of ceramide on its own synthesis was investigated and shown to be inhibitory, providing an explanation that increased ceramide levels and decreased ceramide synthesis inhibit SRE-mediated gene transcription. PlasmidsThe pSyn-SRE luciferase reporter plasmid originates from the hamster HMG-CoA synthase promoter and contains three SRE elements (Ϫ326 to Ϫ225 bp) and has been described before. 14,15 The pWLNeo plasmid was obtained from Stratagene (La Jolla, Calif). Cell Culture and Stable TransfectionsCells were grown in F12-nutrient mixture medium contai...

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