Impaired expression of glutathione synthetic enzyme genes in mice with targeted deletion of the Nrf2 basic-leucine zipper protein

Abstract

Transcriptional activation of genes that play a role in detoxification of xenobiotics and defense against oxidative stress is mediated in part by the antioxidant response element (ARE). For example, it has been shown that the promoters for both the heavy and light chain gamma-glutamylcysteine synthetase (GCS(H) and GCS(L)) genes require the ARE. CNC-bZIP factors, together with small Maf proteins, have been shown to bind as heterodimers to the NF-E2/AP-1 element, which is similar to the consensus sequence for the ARE. Nrf1 and Nrf2, two widely expressed CNC-bZIP factors, have been implicated in the regulation of genes involved in oxidative stress response. In this study, we examined the effect of nrf2 mutation on the expression of genes involved in glutathione synthesis. We observed that transcripts for gcs(H) and gcs(L) genes were decreased in nrf2(-/-) fibroblasts and livers. Correspondingly, glutathione levels were decreased in Nrf2 deficient livers and fibroblasts. By transient transfection studies in nrf2(-/-) fibroblasts, we show that transcriptional activation of reporter constructs bearing the human GCS(L) promoter, as well as the functional ARE of GCS(H) promoter, required the activator protein Nrf2. By electrophoretic mobility shift assay, recombinant Nrf2 binds the ARE of the GCS(L) and GCS(H) promoters. Overexpression of Nrf2 cDNA restored glutathione (GSH) levels in nrf2(-/-) fibroblasts, which correlated with increased steady state levels of gcs(H) and gcs(L) transcripts. These results establish a link between Nrf2 transcription factor and GSH biosynthesis.