DNA sequence determinants of nuclear protein binding to the c-Ha-ras antioxidant/electrophile response element in vascular smooth muscle cells: identification of Nrf2 and heat shock protein 90 beta as heterocomplex components

The antioxidant/electrophile response element (ARE/EpRE) is a cis-acting element involved in redox regulation of c-Ha-ras gene. Protein binding to the ARE/EpRE may be credited to deoxyribonucleic acid sequence; therefore, studies were conducted to evaluate the influence of internal and flanking regions to the 10-bp human c-Ha-ras ARE/EpRE core (hHaras10) on nuclear protein binding in oxidant-treated vascular smooth muscle cells. A protein doublet bound to an extended oligonucleotide comprising the ARE/EpRE core in genomic context (hHaras27), whereas a single complex bound to hHarasl0. Protein binding involved specific interactions of 25- and 23-kDa proteins with hHarasl0, and binding of 80-, 65-, and 55-kDa proteins to hHaras27. Competition assays with hNQO1 and rGSTA2 confirmed the specificity of deoxyribonucleic acid-protein interactions and indicated preferred binding of p25 and p23 to the c-Ha-ras ARE/EpRE. "NNN" sequences within the core afforded unique protein-binding profiles to the c-Ha-ras ARE/EpRE. In addition, Nrf2 and heat shock protein 90beta (p80) were identified as components of the c-Ha-ras ARE/EpRE heterocomplex. We conclude that both internal bases and flanking sequences regulate nuclear protein recruitment and complex assembly on the c-Ha-ras ARE/EpRE.     

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

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.