TCF11/Nrf1 overexpression increases the intracellular glutathione level and can transactivate the Q-glutamylcysteine synthetase (GCS) heavy subunit promoter Mari C.W. Myhrstad a , Cathrine Husberg b , Paula Murphy b , Olov Nordstro «m a , Rune Blomho¡ a , Jan Òyvind Moskaug a , Anne-Brit KolstÖ b ; * a Institute for Nutrition Research, University of Oslo, Oslo, Norway b Biotechnology Centre of Oslo, PB: 1125 Blindern, 0316 Oslo, Norway Received 5 April 2000; received in revised form 27 October 2000; accepted 7 November 2000 Abstract Q-Glutamylcysteinylglycine or glutathione (GSH) performs important protective functions in the cell through maintenance of the intracellular redox balance and elimination of xenobiotics and free radicals. The production of GSH involves a number of enzymes and enzyme subunits offering multiple opportunities for regulation. Two members of the CNC subfamily of bZIP transcription factors (TCF11/ Nrf1 and Nrf2) have been implicated in the regulation of detoxification enzymes and the oxidative stress response. Here we investigate the potential role of one of these factors, TCF11/Nrf1, in the regulation of GSH levels in the cell and particularly its influence on the expression of one of the enzymatic components necessary for the synthesis of GSH, the heavy subunit of Q-glutamylcysteine synthetase (GCS h ). Using overexpression of the transcription factor in COS-1 cells we show that TCF11/Nrf1 stimulates GSH accumulation. Using co-transfection with reporter constructs where reporter expression is driven through the GCS h promoter we show that this increase may be mediated in part by induced expression of the GCS h gene by TCF11/Nrf1. We further show that a distal portion of the promoter including two antioxidant- response elements (AREs) predominantly mediates the TCF11/Nrf1 transactivation and an electromobility shift assay showed that just one of these AREs specifically binds TCF11/Nrf1 as heterodimers with small Maf proteins. We suggest that TCF11/Nrf1 can operate through a subset of AREs to modulate the expression of GCS h together with other components of the pathway and in this way play a role in regulating cellular glutathione levels. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : TCF11/Nrf1 ; Glutathione ; Antioxidant-response element ; Q-Glutamylcysteine synthetase 1. Introduction The tripeptide Q-glutamylcysteinylglycine or glutathione (GSH) is a ubiquitous cellular non-protein sulphydryl. GSH has two important roles in the cell: maintaining the intracellular redox balance and eliminating xenobiotics and free radicals [1]. GSH is synthesised from amino acids in two enzymatic reactions catalysed by Q-glutamylcysteine synthetase (GCS) and glutathione synthetase [2]. The re- action catalysed by GCS is the rate-limiting step in the de novo synthesis of GSH, so that regulation of GCS levels is likely to be an important aspect in the regulation of GSH synthesis [3]. GCS consists of two subunits, the heavy sub- unit (GCS h ) with catalytic activity and the light subunit (GCS l ) with regulatory activity [4^6]. Regulation of en- zyme activity occurs through multiple mechanisms a¡ect- ing one or both subunits and the heavy subunit in partic- ular is regulated by both transcriptional and post- transcriptional mechanisms [7]. Transcriptional control of GCS h is mediated by a region spanning approx. 5 kb of the 5P £anking sequence of the GCS h gene [8]. Analysis of this region by Mulcahy et al. revealed the presence of response elements including sev- eral AP-1 sites, antioxidant-response elements (AREs; also known as the electrophile-response element EpRE) and one NF-UB site. Similar ARE motifs are also found in promoters of other genes that participate in the defence against free radicals and toxic insults, and this has led to the de¢nition of a consensus core ARE motif (A/GTGAC/ GnnnGCA/G). Among such genes are NADPH:quinone oxidoreductase [9], glutathione S-transferase [10] and UDP-glucuronosyltransferase [11]. Many of these genes 0167-4781 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII:S0167-4781(00)00276-1 * Corresponding author. Fax: +47-22-840-501; E-mail : annebko@biotek.uio.no Biochimica et Biophysica Acta 1517 (2001) 212^219 www.elsevier.com/locate/bba