Up-regulation of the human gamma-glutamylcysteine synthetase regulatory subunit gene involves binding of Nrf-2 to an electrophile responsive element

Biochem Biophys Res Commun. 1999 Aug 11;261(3):661-8. doi: 10.1006/bbrc.1999.1109.

Abstract

The rate-limiting step in the de novo synthesis of the cellular protectant glutathione is catalyzed by gamma-glutamylcysteine synthetase (GCS; also known as glutamine-L-cysteine ligase, GLCL), a heterodimer consisting of catalytic (GCS(h)) and regulatory (GCS(l)) subunits. Regulation of expression of the human gamma-glutamylcysteine synthetase regulatory subunit gene in response to beta-NF is mediated by an Electrophile Responsive Element (EpRE) [Moinova, H., and Mulcahy, R. T. (1998) J. Biol. Chem. 273, 14683-14689]. Oligonucleotide probes corresponding to wild-type and mutant EpRE sequences were used in gel-shift and super-shift analyses to identify proteins binding. Four protein:DNA complexes (a-d) with distinct mobilities were detected when the wild-type EpRE probe was incubated with nuclear extracts from control or beta-NF-treated HepG2 cells. Following beta-NF treatment, there was an increase in the intensity of a single band, band b. This band was eliminated in gel shifts employing mutant EpRE probes which abolish beta-NF inducibility, demonstrating a correlation between band b and transactivation. Super-shift analysis identified JunD, Nrf1, and Nrf2 in the EpRE-binding complexes. Antibodies to Nrf2 completely super-shifted the band b protein:DNA complex. These studies demonstrate that Nrf2 proteins recognize and bind the GCS(l) EpRE sequence to affect transactivation of the gene.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Binding Sites
  • Carcinoma, Hepatocellular
  • Cell Nucleus / metabolism
  • DNA / metabolism
  • DNA-Binding Proteins / metabolism*
  • Dimerization
  • GA-Binding Protein Transcription Factor
  • Gene Expression Regulation, Enzymologic*
  • Glutamate-Cysteine Ligase / chemistry
  • Glutamate-Cysteine Ligase / genetics*
  • Glutamate-Cysteine Ligase / metabolism
  • Humans
  • Liver Neoplasms
  • Mutation
  • Regulatory Sequences, Nucleic Acid*
  • Response Elements*
  • Transcription Factors / metabolism*
  • Transcriptional Activation
  • Tumor Cells, Cultured
  • beta-Naphthoflavone / pharmacology

Substances

  • DNA-Binding Proteins
  • GA-Binding Protein Transcription Factor
  • Transcription Factors
  • beta-Naphthoflavone
  • DNA
  • Glutamate-Cysteine Ligase