Induction of glutathione synthesis in macrophages by oxidized low-density lipoproteins is mediated by consensus antioxidant response elements

The uptake of oxidized low-density lipoproteins (oxLDL) by macrophages leading to conversion into foam cells is a seminal event in atherogenesis. Excessive accumulation of oxLDL can cause oxidative stress in foam cells leading to cell death and the progression and destabilization of atherosclerotic lesions. Oxidative stress induces a protective compensatory increase in the synthesis of the endogenous antioxidant glutathione (GSH). Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in GSH synthesis and is composed of a catalytic subunit (GCLC) and a modifier subunit (GCLM), which are products of separate genes. Treatment of RAW 264.7 mouse macrophages and mouse peritoneal macrophages with oxLDL (30 mug/mL) induces increased expression of both Gclc and Gclm in vitro. The increase in mRNA occurs in part via increased transcription as demonstrated with luciferase reporter constructs. The promoters for both GCLC and GCLM contain consensus antioxidant response elements (AREs). Electrophoretic mobility shift assays revealed induction of nuclear factor binding to these AREs after treatment of RAW 264.7 cells and mouse peritoneal macrophages with oxLDL. Nuclear factor binding to the AREs is diminished by a single base pair substitution in the core sequence. Site-directed mutagenesis of the AREs within the Gclc and Gclm promoters resulted in a decrease of oxLDL-induced luciferase activity. Supershift analyses revealed that oxLDL stimulates binding of the transcription factors Nrf1, Nrf2, and c-jun to the AREs. These data suggest that AREs play a direct role in mediating the induction of GSH synthesis by oxLDL and in protecting macrophages against oxidized lipid-induced oxidative stress.