The non-protein thiol, glutathione (gamma-glutamyl-cysteinyl-glycine, GSH) is a predominant cellular antioxidant and as such serves critical functions in the maintenance of cellular redox balance, provides protection against reactive oxygen species and is involved in the detoxication of xenobiotics either through direct reactions with reactive intermediates or via enzymatic conjugation reactions catalyzed by glutathione S-transferases. Exposure of cells to a number of xenobiotic agents results in a significant increase in the total intracellular GSH content, secondary to transcriptional up-regulation of the genes encoding the two protein subunits (catalytic and regulatory) of gamma-glutamylcysteine synthetase (GCS, EC 6.3.2.2), the rate-limiting enzyme in its de novo synthesis. It is hypothesized that transcriptional up-regulation of the two GCS subunit genes involves similar cis-elements, but distinct combinations of trans- factors, contributing to differential regulation in response to specific inducting agents. Transcription is hypothesized to involve dimeric transcription factors composed of small Maf proteins and various other bZIP family members, including Nrf1, Nrf2, Fos and Jun. Furthermore, transcriptional activation is hypothesized to be mediated by specific MAPK signaling pathways in response to alterations in the cellular redox balance in favor of a more pro-oxidant state. In evaluating these hypotheses, we propose the following Specific Aims: 1. Finalize analysis of cis- elements within the heavy and light subunit promoters to identify those involved in transcriptional activation of the genes in response to beta-NF; tBOOH; menadione, H202 and PDTC. 2. Identify the transactivating factors and their component proteins which are ultimately involved in binding to the specific cis- elements identified in Aim 1. 3. Determine the role that oxidative stress plays in GCS subunit gene induction. 4. Define the signaling pathway(s) involved in up-regulation of GCS subunit genes. The application proposes a comprehensive investigation of the nature of the signals, the signaling pathways, and the trans- and cis- factors which in composite constitute the mechanism of GCS gene regulation under constitutive conditions and in response to the selective classes of agents included in the investigation.
