Identification and characterization of an Nrf2-mediated ARE upstream of the rat glutamate cysteine ligase catalytic subunit gene (GCLC).
The antioxidant response element (ARE) is an essential component of upstream regulatory sequences present on genes for most phase II detoxification enzymes, including the glutamate cysteine ligase catalytic subunit (GCLC). NF-E2-related factor 2 (Nrf2) is a principal transcription factor that binds to the ARE and plays a key role in cellular responses to stress via the Keap1-Nrf2-ARE pathway. However, the ARE that mediates human GCLC gene expression has not been found in the rat. Thus, how the ARE-mediated Keap1-Nrf2-ARE pathway regulates glutathione homeostasis in the rat remains a puzzle. We have identified a putative ARE sequence approximately 4 kb upstream in the rat GCLC. We further defined the rat GCLC-ARE in the category with the most ARE characters, that is, this rat GCLC-ARE is a sequence-specific site that significantly enhances promoter activity in reporter genes. The rat GCLC-ARE is an Nrf2-mediated element to which binding has been demonstrated in nuclear extracts and induced by tert-butylhydroquinone. Given the central role that rat models play in toxicology and pathology, this first discovery of the rat GCLC-ARE enhancer similar to that found in the human gene has broad implications for the study of antioxidant defenses and their regulation in a number of different fields.
Li
Muyao
M
Department of Medicine, University of Vermont College of Medicine, Burlington, Vermont 05405, USA.
Chiu
Jen-Fu
JF
Kelsen
Anne
A
Lu
Shelly C
SC
Fukagawa
Naomi K
NK
D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics*; D047369:Chromatin Immunoprecipitation; D017930:Genes, Reporter; D005721:Glutamate-Cysteine Ligase / Q000235:genetics*; D006873:Hydroquinones / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D051267:NF-E2-Related Factor 2 / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D011916:Rats, Inbred F344; D020218:Response Elements / Q000235:genetics*
D006873:Hydroquinones; D051267:NF-E2-Related Factor 2; C495637:Nfe2l2 protein, rat; D009687:Nuclear Proteins; C018855:2-tert-butylhydroquinone; D008156:Luciferases; D005721:Glutamate-Cysteine Ligase; C544143:GCLC protein, rat