Activation of mouse Pi-class glutathione S-transferase gene by Nrf2(NF-E2-related factor 2) and androgen

Abstract

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 500 bp 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.

Identification of a Functional Antioxidant Response Element within the Eighth Intron of the HumanABCC3Gene

“…Other examples of nuclear receptors with identified response elements within an intronic region include the androgen receptor (Zheng et al, 2006), the vitamin D receptor (Zella et al, 2006), and peroxisome proliferator-activated receptora (Sun et al, 2008). In addition, androgen regulation of the Nrf2-target gene GSTP1 appears to be dependent upon a 500-bp region of the fifth intron of the gene (Ikeda et al, 2002); however, to our knowledge, this is the first example of Nrf2 itself potentially regulating a target gene through an intronic response element.…”

Haplotype-Environment Interactions That Regulate the Human Glutathione S-Transferase P1 Promoter

“…The roles of Keap1, Nrf2, and the ARE in phase II transcriptional events have been extensively explored in certain models (40)(41)(42). Whereas BITC and sulforaphane are known to act through the Nrf2 binding to ARE, three ARE elements exist in the mouse GSTP1 promoter, which is thought similar to the human GSTP1 promoter (38,43). Therefore, the details of interspecies and intertissue differences in the isothiocyanate-modulated GSTP1 transcriptional cascades remain unclear.…”

Aldehyde oxidase 1 gene is regulated by Nrf2 pathway

“…This is an interesting observation since most of the Nrf2 target genes have a single ARE in its promoter (Ikeda et al, 2004;Ikeda et al, 2002;Jaiswal, 2000;Rushmore et al, 1991), which is sufficient for Nrf2 activation. It remains unclear why AOX1 gene requires two AREs for the Nrf2 responses.…”

“…For the reporter transfection assay, a total of 0.6µg DNA (0.2 µg of reporter plasmid, 0.2 µg of the β-galactosidase expression plasmid (pSVβgal, Promega) and 0.2 µg of the Nrf2 expression plasmid (Ikeda et al, 2002) or empty expression vector (pcDNAver.3.1, Invitrogen)) were transfected into cells using the transfection agent FuGENE (Roche Applied Science) according to the supplier's protocol. At 45 hr post-transfection, cells were harvested and assayed for luciferase activity using a luciferase assay kit (Nippon Gene, Japan) and for β-galactosidase activity.…”

“…Small Maf proteins lack the transactivation domain present in the large Maf proteins (i.e., in c-Maf, MafB, NRL, and LMaf/MafA). The Nrf2/small Maf heterodimer binds to ARE (anti-oxidant responsive element) and to MARE (Maf recognition element), and regulates downstream genes, including those of phase II detoxifying enzymes (Ikeda et al, 2004;Ikeda et al, 2002;Jaiswal, 2000;Nioi et al, 2003). protein) as a key mediator for the induction of phase II detoxifying enzymes by electrophilic drugs (Itoh et al, 1999;Wakabayashi et al, 2003).…”