Regulation of gamma-glutamylcysteine synthetase regulatory subunit (GLCLR)gene expression: Identification of the major transcriptional start site inHT29 cells

gamma-Glutamylcysteine synthetase (GCS) is of major importance in glutathio ne homeostasis. The GCS heterodimer is composed of catalytic (heavy subunit , GCS(h)) and regulatory (light subunit, GCS(l)) subunits. Regulation of th e human GCS(l) subunit gene (GLCLR) expression was studied as GCS(l) has a critical role in glutathione synthesis. The minimal basal expression of GLC LR was found to be mediated by a region between nt -205 and -318. The major transcriptional start site in HT29 cells was located within this region at nt -283. A region between nt -411 and -447 was identified as having a pote ntial involvement in the negative regulation of GLCLR expression. In order to study the transcriptional regulation of GCS(l) by oxidant stress, HepG2 cells were treated with sodium nitroprusside (SNP). SNP (1.5 mM) was found to increase glutathione levels by 2-fold, as well as GCS activity by 6-fold . This is accompanied by a co-ordinate increase in the levels of the both t he GCS(l) and GCSh subunits, each by approximately 2-fold. The transcriptio nal activity of the GLCLR gene was increased by approximately 2.5-fold in S NP-treated cells. (C) 1999 Elsevier Science B.V. All rights reserved.