The Pi-class glutathione S-transferases (GSTs) play pivotal roles in the detoxification of xenobiotics, carcinogenesis and drug resistance. The mechanisms of regulation of these genes during drug induction and carcinogenesis are yet to be elucidated. Recently, Nrf2 (NF-E2-related factor 2; a bZip-type transcription factor) knockout mice were shown to display impaired induction of Pi-class GST genes by drugs. It is known that the mouse Pi-class GST gene GST-P1 is expressed predominantly in the male liver, and is regulated by androgen. To determine whether Nrf2 and the androgen receptor regulate GST-P1 directly, we analysed the molecular mechanism of activation of this gene by these factors. The promoter of the GST-P1 gene was activated markedly by Nrf2 in transient transfection analyses. Gel mobility shift assay and footprinting analyses revealed three Nrf2 binding sites: one at the proximal and two at distal elements, located at positions −59, −915 and −937 from the cap site. The fifth intron of the GST-P1 gene contains the androgen-responsive region. Multiple androgen receptor binding sites are clustered within a 500bp region of this intron. The whole fragment contains a minimum of seven androgen receptor binding sites, which collectively display strong androgen-dependent enhancer activity. However, on division into small fragments containing two or three elements each, individual enhancer activities were dramatically decreased. This suggests that multiple elements work synergistically as a strong androgen-responsive enhancer. Our findings indicate that Nrf2 and the androgen receptor directly bind to and activate the mouse GST-P1 gene.
Abbreviations used: ARE, antioxidant-responsive element; EMSA, electrophoretic mobility shift assay; GPE1, strong enhancer element of the rat GST-P gene; GST, glutathione S-transferase; GST-P1, mouse Pi-class GST; MBP, maltose binding protein; NQO1, NAD(P)H:quinone oxidoreductase; Nrf, NF-E2-related factor; RT-PCR, reverse transcription—PCR; TRE, PMA ('TPA')-responsive element.
The nucleotide and predicted amino acid sequence of rat Nrf2 cDNA are registered at the GenBank®, EMBL, DDBJ and GSDB Nucleotide Sequence Databases under accession no. AF037350.