Nε-(Carboxymethyl)lysine induces γ-glutamylcysteine synthetase in RAW264.7 cells

Abstract

Advanced glycation end products (AGEs) play an important role in the development of angiopathy in diabetes mellitus and atherosclerosis. Here, we show that adducts of N ε-(carboxymethyl)lysine (CML), a major AGE, and bovine serum albumin (CML-BSA) stimulated gamma-glutamylcysteine synthetase (γ-GCS), which is a key enzyme of glutathione (GSH) synthesis, in RAW264.7 mouse macrophage-like cells. CML-BSA stimulated the expression of γ-GCS heavy subunit (h) time- and dose-dependently and concomitantly increased GSH levels. CML-BSA also stimulated DNA-binding activity of activator protein-1 (AP-1) within 3 h, but the stimulatory effect decreased in 5 h, and nuclear factor-κB (NF-κB) with a peak activity at 1 h and the stimulatory effect diminished in 3 h. Studies of luciferase activity of the γ-GCSh promoter showed that deletion and mutagenesis of the AP-1-site abolished CML-BSA-induced up-regulation, while that of NF-κB-site did not affect CML-BSA-induced activity. CML-BSA also stimulated the activity of protein kinase C, Ras/Raf-1, and MEK/ERK1/2. Inhibition of ERK1/2 abolished CML-BSA-stimulated AP-1 DNA-binding activity and γ-GCSh mRNA expression. Our results suggest that induction of γ-GCS by CML adducts seems to increase the defense potential of cells against oxidative stress produced during glycation processes.