Regulation of gamma-glutamylcysteine synthetase subunit gene expression bythe transcription factor Nrf2

Exposure of HepG2 cells to beta-naphthoflavone (beta-NF) or pyrrolidine dit hiocarbamate (PDTC) resulted in the up-regulation of the gamma-glutamylcyst eine synthetase catalytic (GCS(h)) and regulatory (GCS(1)) subunit genes. I ncreased expression was associated with an increase in the binding of Nrf2 to electrophile response elements (EpRE) in the promoters of these genes. N rf2 overexpression increased the activity of GCS(h) and GCS(1) promoter/rep orter transgenes, Overexpression of an MafK dominant negative mutant decrea sed Nrf2 binding to GCS EpRE sequences, inhibited the inducible expression of GCS, and GCS, promoter/reporter transgenes, and reduced endogenous GCS g ene induction. beta-NF and PDTC exposure also increased steady-state levels of MafG mRNA, In addition to Nrf2, small Maf and JunD proteins were detect ed in GCS(h)EpRE-protein complexes and, to a lesser extent, in GCS(1)EpRE-p rotein complexes. The Nrf2-associated expression of GCS promoter/reporter t ransgenes was inhibited by overexpression of MafG, Inhibition of protein sy nthesis by cycloheximide partially decreased inducibility by PDTC or beta-N F and resulted in significant increases in GCS mRNA at late time points, wh en GCS mRNA levels are normally declining, We hypothesize that, in response to beta-NF and PDTC, the GCS subunit genes are transcriptionally upregulat ed by Nrf2-basic leucine zipper complexes, containing either JunD or small Maf protein, depending on the particular GCS EpRE target sequence and the i nducer. Following maximal induction, down-regulation of the two genes is me diated via a protein synthesis-dependent mechanism.