Antioxidant Defense: Effects of an NRF2 ARE Polymorphism on the Activation of Phase II Enzymes

Abstract

The purpose of the research put forth in this thesis has been to critically evaluate the functional effects of a single nucleotide polymorphism in the antioxidant response element (ARE) of NRF2. NRF2 is a transcription factor that regulates numerous antioxidants in response to oxidative stress, and we believe mutation within the ARE may impact both transcription and translation of downstream antioxidants. Initial studies focus on quantitative differences in NRF2 expression between ARE clones with and without mutation using transient transfection assays as well as diminished NRF2:ARE binding by gel shift analysis. Effects of decreased function are further analyzed using lymphoblast cell lines with naturally occurring NRF2 ARE mutations (wildtype, ARE heterozygote and ARE variant) exposed to oxidative stress. Differences in mRNA expression of NRF2 and downstream antioxidants are assessed by reverse transcriptase PCR and quantitative real-time PCR, and protein level changes are analyzed by western blot.

These studies show that NRF2 function is decreased with mutation to the ARE and NRF2:ARE binding is diminished in the presence of variant alleles. Furthermore, transcription of NRF2 and several downstream antioxidants (NQO1, HO-1 and GPx2) is significantly upregulated in heterozygotes with one copy of the variant allele during serum starvation and following exposure to several oxidative stress agents [hyperoxia (95% oxygen) and lipopolysaccharide]. However, increases in mRNA transcripts do not directly correlate with protein levels. Nonetheless, we did observe differential protein expression between NRF2 genotypes for NRF2, the accessory protein small Maf, as well as ARE-bearing downstream antioxidants (NQO1, HO-1, GPx2 and GSR). The consequences of these functional differences are likely to impact the cellular response to oxidative stress.