Glutathione (GSH) plays a vital defensive role and modulates critical cellular processes. One important factor that determines the rate of GSH synthesis is the activity of glutamate-cysteine ligase (GCL, also known as glutamylcysteine synthetase). GCL is made up of a catalytic and a modifier subunit (GCLC and GCLM), the former exhibits all of the catalytic activity of the holoenzyme but the latter makes the enzyme function more efficiently. During the reporting period we showed that the two subunits are differentially regulated. While hormones and rapid liver growth transcriptionally activate GCLC, some but not all inducers of oxidative stress activate both GCL subunits. Although much emphasis has been placed on GCL, our results indicate that the second enzyme in GSH synthesis, GSH synthetase (GS) may be just as important in certain cells and has been overlooked. Treatments that induce both GCL subunits also induced GS. Importantly, we found GS up-regulation can further enhance the cell's GSH synthetic capacity. In order to study transcriptional regulation of the GSH synthetic enzymes using our well-defined in vivo and in vitro models, we have cloned the 5'-flanking regions of both GCL subunits and GS from the rat and identified candidate transcription factors that may regulate these genes. We found AP-1 is required for the basal expression and the tert-butylhydroquinone-mediated increase in expression of both GCL subunits and GS. The advantage of studying the rat promoters is the ability to perform comparative studies using in vitro and in vivo models, which would be difficult to achieve with human promoters. Finally, we characterized GSH homeostasis in regenerating rat liver and human hepatocellular carcinoma (HCC) and found that both GCLC and GS are transcriptionally up-regulated in HCC and increased GSH can facilitate liver cancer cell growth. The following aims are direct extensions of these observations which will: 1) examine transcriptional regulation of rat GCLC - define the molecular mechanism of transcriptional regulation by various agents we have identified using in vitro and in vivo models; 2) examine transcriptional regulation of rat GCLM - similar types of studies will be performed here as for GCLC; and 3) examine transcriptional regulation of rat GS - identify cis-acting elements and transcription factors important for basal expression and in response to various treatments. These studies should improve our understanding of transcriptional regulation of the GSH synthetic enzymes. Our ultimate goal is to utilize this information to improve the treatment and prevent complications that may result from altered hepatic GSH synthesis. ? ?
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