TCF11/Nrf1 overexpression increases the intracellular glutathione level and can transactivate the gamma-glutamylcysteine synthetase (GCS) heavy subunit promoter

gamma -Glutamylcysteinylglycine or glutathione (GSH) performs important pro tective functions in the cell through maintenance of the intracellular redo x balance and elimination of xenobiotics and free radicals. The production of GSH involves a number of enzymes and enzyme subunits offering multiple o pportunities for regulation. Two members of the CNC subfamily of bZIP trans cription factors (TCF11/ Nrf1 and Nrf2) have been implicated in the regulat ion of detoxification enzymes and the oxidative stress response. Here we in vestigate the potential role of one of these factors, TCF11/Nrf1, in the re gulation of GSH levels in the cell and particularly its influence on the ex pression of one of the enzymatic components necessary for the synthesis of GSH, the heavy subunit of gamma -glutamylcysteine synthetase (GCS(h)). Usin g overexpression of the transcription factor in COS-1 cells we show that TC F11/Nrf1 stimulates GSH accumulation. Using co-transfection with reporter c onstructs where reporter expression is driven through the GCSh promoter we show that this increase may be mediated in part by induced expression of th e GCSh gene by tCF11/Nrf1. We further show that a distal portion of the pro moter including two antioxidant-response elements (AREs) predominantly medi ates the TCF11/Nrf1 transactivation and an electromobility shift assay show ed that just one of these AREs specifically binds TCF11/Nrf1 as heterodimer s with small Maf proteins. We suggest that TCF11/Nrf1 can operate through a subset of AREs to modulate the expression of GCSh together with other comp onents of the pathway and in this way play a role in regulating cellular gl utathione levels. (C) 2001 Elsevier Science B.V. All rights reserved.