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Hyperhomocysteinemia is an independent risk factor for vascular disease. However, the mechanisms by which hyperhomocysteinemia promotes the development of atherosclerosis are unknown. The accumulation of macrophages in the vascular wall is an early step in the development of atherosclerosis. Uptake of homocysteine induces oxidative stress in macrophages. Antioxidant response elements (ARE) are regulatory elements within promoters of genes, which generally function to protect cells against oxidative stress. In the current study, we investigated whether homocysteine induces transcriptional activity of AREs and ARE driven gene expression of the enzyme glutamate-cysteine ligase (GCL) in macrophages. GCL is the rate-limiting enzyme in the synthesis of glutathione, an important endogenous antioxidant, and contains several AREs within its promoter. Treatment of mouse macrophages with homocysteine in a concentration, which is found in plasma of hyperhomocysteinemic individuals (50 μg/ml), induced increased binding of nuclear protein to the AREs. Real-time RT-PCR demonstrated increased RNA-expression of GCL after treatment with homocysteine. The increase in mRNA occured via increased transcription as demonstrated with luciferase promoter reporter constructs for GCL. Inhibition of the MAP kinase activity reduced binding of nuclear proteins to the AREs and mRNA expression of GCL. Western blotting demonstrated phosporylation of ERK1/2, but not p38, in homocysteine treated macrophages. Additional site directed mutagenesis studies are currently employed to determine whether AREs play a direct role in mediating induction of GCL by homocysteine in macrophages.
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