c-Maf negatively regulates ARE-mediated detoxifying enzyme genes expression and anti-oxidant induction

Abstract

Anti-oxidant response element (ARE) and nuclear factors including Nrf2 and small Maf (MafG and MafK) proteins are known to regulate expression and induction of detoxifying enzyme genes including quinone oxidoreductase1 (NQO1). Nrf2 upregulates and small Maf proteins lacking the transcriptional activation domain down regulates ARE-mediated expression and induction. In this report, we have investigated the role of c-Maf (large Maf) containing the transcriptional activation domain in the regulation of ARE-mediated genes expression. The overexpression of c-Maf in human hepatoblastoma (Hep-G2) cells led to the repression of ARE-mediated NQO1 and GST Ya genes expression and induction in response to tert-butyl hydroquinone (t-BHQ). This was in contrast to the role of c-Maf in the activation of Maf recognition element (MARE) mediated p53 gene expression. Deletion of transcriptional activation domain of c-Maf (ĉ-Maf) led to significant loss of MARE-mediated p53 gene expression but had no effect on the repression of ARE-mediated NQO1 gene expression. The overexpression of MafG in Hep-G2 cells repressed both ARE and MARE-mediated genes expression. The co-expression of c-Maf with MafG rescued the MafG repression of MARE but not ARE-mediated gene expression. Band and super shift assays showed the presence of c-Maf in the ARE-nuclear protein complex. Similar assays with in vitro translated proteins revealed that both c-Maf and ĉ-Maf bound to NQO1 gene ARE as homodimers and heterodimers with small Maf but not as heterodimers with Nrf2. Mutational analysis of the NQO1 gene ARE indicated that core ARE sequence is essential for binding of c-Maf leading to repression of NQO1 gene expression. Northern analysis revealed that c-Maf expression increases 2 h after t-BHQ treatment. It reached a plateau at 4 h after t-BHQ treatment. The results together led to the conclusion that c-Maf negatively regulates ARE-mediated detoxifying enzyme genes expression and induction in response to anti-oxidants.