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.