Abstract
Despite it being a quintessential Phase II detoxification gene, the transcriptional regulation of the rat gamma-glutamate cysteine ligase catalytic subunit (GCLC) is controversial. Computer-based sequence analysis identified three putative antioxidant response elements (AREs) at positions -889 to -865 (ARE1), -3170 to -3146 (ARE2) and -3901 to -3877 (ARE3) in the 5'-flanking region of the transcriptional start site. Transfections of individual ARE-luciferase reporter gene constructs into H4IIE cells, a rat hepatoma cell line, identified ARE3 as the functional promoter. Chromatin immunoprecipitation assays using primary rat hepatocytes showed that the transcription factor Nrf2, which is known to regulate ARE-mediated genes, is associated with ARE3. Co-transfection of H4IIE cells with luciferase reporter plasmids containing Gclc ARE3 and an Nrf2 expression plasmid resulted in a 3-fold activation of ARE3-mediated transcription relative to controls. "Loss-of-function" analysis for Nrf2 by small interfering RNA (siRNA) revealed that ARE3-mediated expression was significantly impaired while site-directed mutagenesis of the ARE3-luciferase reporter abolished Nrf2-mediated induction. Treatment with two known Nrf2 inducers, R-(alpha)-lipoic acid and anetholedithiolethione, showed that the inducible expression of the GCLC gene was also regulated by the ARE3 element. Taken together, these results show that Nrf2 regulates the constitutive expression of rat Gclc through a distal ARE present in its 5'-flanking region. This is the first report showing that rat Gclc is under the transcriptional control of the Nrf2-ARE pathway on a constitutive basis.