Dual effect of ceramide on human endothelial cells: induction of oxidative stress and transcriptional upregulation of endothelial nitric oxide synthase.
BACKGROUND
Generation of the second-messenger molecule ceramide by stimulated sphingomyelinase activity has been implicated in the inflammatory processes contributing to the pathogenesis of atherosclerosis. However, reports of stimulatory effects of ceramide on endothelial NO production in animal models suggest antiatherosclerotic effects of the molecule. Therefore, we investigated long-term effects of ceramide on NO generation in human endothelial cells.


RESULTS
In human umbilical vein endothelial cells (HUVECs) and in HUVEC-derived EA.hy 926 endothelial cells, C6-ceramide (N-hexanoyl-D-erythro-sphingosine) reduced the generation of bioactive NO (RFL-6 reporter-cell assay). At the same time, the signaling molecule increased endothelial NO synthase (eNOS) mRNA (RNase protection assay) and protein expression (Western blot). C6-ceramide stimulated eNOS transcription by a signaling mechanism involving protein phosphatase PP2A but did not modify the stability of the eNOS mRNA. Endothelial generation of reactive oxygen species (ROS) was increased by C6-ceramide [5-(and-6)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA) oxidation-based fluorescence assay], and this effect was partially reversed by the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). On the other hand, (6R)-5,6,7,8-tetrahydro-L-biopterin (BH(4)) normalized in part the ceramide-induced reduction in bioactive NO.


CONCLUSIONS
Ceramide produces oxidative stress in human endothelial cells, thereby reducing bioactive NO. The partial reversal of this reduction by BH(4) and the diminution of ROS generation by L-NAME suggest that ceramide promotes NADPH oxidase activity of eNOS, leading to ROS formation at the expense of NO synthesis. The ceramide-induced upregulation of eNOS gene transcription can be considered an ineffective compensatory mechanism. The decreased bioavailability of NO is likely to favor a proatherogenic role of ceramide.
Li
Huige
H
Department of Pharmacology, Johannes Gutenberg University, Mainz, Germany.
Junk
Peter
P
Huwiler
Andrea
A
Burkhardt
Christian
C
Wallerath
Thomas
T
Pfeilschifter
Josef
J
Förstermann
Ulrich
U
D002460:Cell Line; D002478:Cells, Cultured; D002518:Ceramides / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000235:genetics*; D052250:Nitric Oxide Synthase Type III; D018384:Oxidative Stress; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D011401:Promoter Regions, Genetic; D020871:RNA Stability; D017382:Reactive Oxygen Species / Q000378:metabolism; D013108:Sphingomyelin Phosphodiesterase / Q000494:pharmacology; D015533:Transcriptional Activation; D015854:Up-Regulation
D002518:Ceramides; D004791:Enzyme Inhibitors; D017382:Reactive Oxygen Species; C101954:N-caproylsphingosine; D009569:Nitric Oxide; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D010749:Phosphoprotein Phosphatases; D013108:Sphingomyelin Phosphodiesterase