Homocysteine decreases endothelin-1 expression by interfering with the AP-1 signaling pathway.
The increased cardiovascular risk associated with hyperhomocysteinemia has been linked to homocysteine-induced endothelial cell (EC) dysfunction. Endothelin-1 is a vasoactive peptide, synthesized mainly by vascular ECs. We have previously shown that homocysteine decreases endothelin-1 biosynthesis. Here we addressed the molecular mechanism of endothelin-1 regulation by homocysteine. Experiments with the transcription inhibitor actinomycin D indicated that the decrease in preproendothelin-1 mRNA content in homocysteine-treated cells did not result from transcript destabilization. Transient transfection assays demonstrated that homocysteine downregulated endothelin-1 at the transcriptional level by decreasing preproendothelin-1 promoter activity. Mutation of the activator protein-1 (AP-1) site of the promoter eliminated the repression induced by homocysteine. Western blot analysis showed that the homocysteine-induced decrease in promoter activity was not associated with reduced expression of the AP-1 components c-Fos and c-Jun. The inhibitory action of homocysteine on preproendothelin-1 mRNA expression was not prevented by cycloheximide. Electrophoretic mobility shift assays demonstrated that homocysteine reduced the binding activity of ECs nuclear extracts to an AP-1 consensus site. These results indicate that homocysteine downregulates endothelin-1 synthesis by inhibiting AP-1 activity, and that the AP-1 signaling pathway may be of major importance in homocysteine-induced endothelial dysfunction.
Laboratoire de Biochimie Appliquée and IFR ISIT, Faculté de Pharmacie, Université Paris XI, Chatenay-Malabry, France.
Séverine
S
Drunat
Nicole
N
Moatti
Karine
K
Demuth
D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D015536:Down-Regulation; D019332:Endothelin-1 / Q000096:biosynthesis*; D004727:Endothelium / Q000166:cytology; D006710:Homocysteine / Q000378:metabolism*; D006801:Humans; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D019332:Endothelin-1; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D018808:Transcription Factor AP-1; D006710:Homocysteine; D003513:Cycloheximide