n-caproylsphingosine and sphingosine-kinase

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.. Experiments were performed in Chinese hamster ovary cells. Inhibition of ceramide synthesis with myriocin, cycloserine, or fumonisin decreases levels of transcriptionally active SREBP and reduces SRE-mediated gene transcription. When ceramide synthesis is increased through exogenous sphingosine or inhibition of sphingosine kinase, SRE-mediated gene transcription is increased. The important role of ceramide synthesis in SRE-mediated gene transcription is confirmed in LY-B cells that do not synthesize ceramide de novo. LY-B cells fail to increase SRE-mediated gene transcription in sterol depletion.. Ceramide synthesis correlates with the generation of transcriptionally active SREBP and SRE-mediated gene transcription. Inhibition of ceramide synthesis decreases levels of transcriptionally active SREBP and SRE-mediated gene transcription. It is hypothesized that the process of ongoing ceramide synthesis contributes to the physiological processing of SREBP, perhaps affecting ER-to-Golgi trafficking. Taken together, modification of ceramide synthesis could be a novel target for drug development in the pharmacologic modification of SRE-dependent pathways.

Topics: 1-Deoxynojirimycin; Acyltransferases; Amidohydrolases; Animals; CCAAT-Enhancer-Binding Proteins; Ceramides; CHO Cells; Cholesterol; Cricetinae; Cricetulus; Cycloserine; DNA-Binding Proteins; Enzyme Induction; Fatty Acids, Monounsaturated; Fumonisins; Genes, Reporter; Hydroxymethylglutaryl-CoA Synthase; Morpholines; Myristates; Phosphotransferases (Alcohol Group Acceptor); Propanolamines; RNA Processing, Post-Transcriptional; Serine C-Palmitoyltransferase; Sphingolipids; Sphingosine; Sterol Regulatory Element Binding Protein 1; Transcription Factors; Transcription, Genetic; Transfection

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that plays a critical role in tissue repair and fibrosis. Sphingolipid signaling has been shown to regulate a variety of cellular processes and has been implicated in collagen gene regulation. The present study was undertaken to determine whether endogenous sphingolipids are involved in the TGF-beta signaling pathway. TGF-beta treatment induced endogenous ceramide levels in a time-dependent manner within 5-15 min of cell stimulation. Using human fibroblasts transfected with a alpha2(I) collagen promoter/reporter gene construct (COL1A2), C(6)-ceramide (10 microm) exerted a stimulatory effect on basal and TGF-beta-induced activity of this promoter. Next, to define the effects of endogenous sphingolipids on TGF-beta signaling we employed ectopic expression of enzymes involved in sphingolipid metabolism. Sphingosine 1-phosphate phosphatase (YSR2) stimulated basal COL1A2 promoter activity and cooperated with TGF-beta in activation of this promoter. Furthermore, overexpression of YSR2 resulted in the pronounced increase of COL1A1 and COL1A2 mRNA levels. Conversely, overexpression of sphingosine kinase (SPHK1) inhibited basal and TGF-beta-stimulated COL1A2 promoter activity. These results suggest that endogenous ceramide, but not sphingosine or sphingosine 1-phosphate, is a positive regulator of collagen gene expression. Mechanistically, we demonstrate that Smad3 is a target of YSR2. TGF-beta-induced Smad3 phosphorylation was elevated in the presence of YSR2. Cotransfection of YSR2 with wild-type Smad3, but not with the phosphorylation-deficient mutant of Smad3 (Smad3A), resulted in a dramatic increase of COL1A2 promoter activity. In conclusion, this study demonstrates a direct role for the endogenous sphingolipid mediators in regulating the TGF-beta signaling pathway.

Topics: Adenoviridae; Ceramides; Collagen; Collagen Type I; DNA-Binding Proteins; Dose-Response Relationship, Drug; Fibroblasts; Humans; Immunoblotting; Kinetics; Luciferases; Membrane Proteins; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Plasmids; Precipitin Tests; Promoter Regions, Genetic; RNA, Messenger; Signal Transduction; Smad3 Protein; Sphingolipids; Time Factors; Trans-Activators; Transfection; Transforming Growth Factor beta