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 90β as heterocomplex components

Abstract

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 hHaras10. Protein binding involved specific interactions of 25-and 23-kDa proteins with hHaras10, and binding of 80-, 65-, and 55-kDa proteins to hHaras27. Competition assays with hNQO 1 and rGSTA2 confirmed the specificity of deoxyribonucleic acid-protein interactions and indicated preferred binding of p25 and p23 to the cHa-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 90␤ (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.

Context‐specific regulation of LINE‐1

“…(Chen & Ramos 2000). AHR functions as a transcriptional regulator of genes involved in bioactivation of BaP to intermediates that cause DNA damage and oxidative stress (Kerzee & Ramos 2000), and bind to redox-regulated elements in BaP target genes (Miller & Ramos 2005). A role for AHR in the regulation of L1 is consistent with the ability of TCDD, a potent activator of AHR, to induce L1 in HeLa cells.…”

NRF2 activation with Protandim attenuates salt‐induced vascular dysfunction and microvascular rarefaction

“…The role of the NRF2/Keap1 system (expressed in endothelial cells and in vascular smooth muscle cells) in cardiovascular homeostasis is still emerging. A role for NRF2 in regulating endothelium‐dependent vasodilation in the human vasculature is suggested by the results of a study by Marczak et al who reported that the ‐653G polymorphism within the Nrf2 promoter region is associated with reduced forearm blood flow and increased forearm vascular resistance in healthy African American volunteers .…”

“…The role of the NRF2/Keap1 system (expressed in endothelial cells 32 and in vascular smooth muscle cells 33,34 ) in cardiovascular homeostasis is still emerging. A role for NRF2 in regulating endothelium-dependent vasodilation in the human vasculature is suggested by the NO-dependent, as it was eliminated when the vessels were acutely exposed to the eNOS inhibitor L-NAME.…”

Lipid aldehydes are substrates and transcriptional regulators of cytochromes P450.

“…The activity of the AP-l transcription factors is regulated by components 01' By performing point mutation studies, it was determined that the "OC" in the ARF core sequence is an absolute requirement of a functional ARE (Rushmore e/ ill., 199 J). It l~as also heen demonstrated in the human c-Ha-ras gene that in addition to the nanking sequence, the trinucloetide "NNN" within the core ARE also intluenees the recruitment of proteins to the ARE (Miller and Ramos, 2005).…”