Abstract
The transcription factor NRF2 (NFE2L2) is a pivotal activator of genes encoding cytoprotective and detoxifying enzymes that limit the action of cytotoxic therapies in cancer. NRF2 acts by binding antioxidant response elements (ARE) in its target genes, but there is relatively limited knowledge about how it is negatively controlled. Here, we report that retinoic X receptor alpha (RXRα) is a hitherto unrecognized repressor of NRF2. RNAi-mediated knockdown of RXRα increased basal ARE-driven gene expression and induction of ARE-driven genes by the NRF2 activator tert-butylhydroquinone (tBHQ). Conversely, overexpression of RXRα decreased ARE-driven gene expression. Biochemical investigations showed that RXRα interacts physically with NRF2 in cancer cells and in murine small intestine and liver tissues. Furthermore, RXRα bound to ARE sequences in the promoters of NRF2-regulated genes. RXRα loading onto AREs was concomitant with the presence of NRF2, supporting the hypothesis that a direct interaction between the two proteins on gene promoters accounts for the antagonism of ARE-driven gene expression. Mutation analyses revealed that interaction between the two transcription factors involves the DNA-binding domain of RXRα and a region comprising amino acids 209-316 in human NRF2 that had not been defined functionally, but that we now designate as the NRF2-ECH homology (Neh) 7 domain. In non-small cell lung cancer cells where NRF2 levels are elevated, RXRα expression downregulated NRF2 and sensitized cells to the cytotoxic effects of therapeutic drugs. In summary, our findings show that RXRα diminishes cytoprotection by NRF2 by binding directly to the newly defined Neh7 domain in NRF2.
Highlights
The human body is continuously threatened by reactive oxygen species (ROS) and electrophiles that are generated by metabolism and by environmental agents
In MCF7 cells cotransfected with the antioxidant response element (ARE)-driven reporter plasmid pGL-GSTA2.41bp-ARE, knockdown of retinoic X receptor alpha (RXRa) was found to increase basal luciferase reporter activity about 1.5-fold, and increased induction of the reporter gene activity by 20 mmol/L tert-butylhydroquinone from 4- to 6-fold
Knockdown of RXRa in Caco2 cells resulted in a 2-fold increase in the basal levels of mRNA for endogenous AKR1C1 and HO-1, both of which are NF-E2 p45related factor 2 (NRF2)-target genes
Summary
The human body is continuously threatened by reactive oxygen species (ROS) and electrophiles that are generated by metabolism and by environmental agents. The NF-E2 p45related factor 2 (NRF2) is a cap'n'collar (CNC) basic-region leucine zipper (bZIP) transcription factor, which plays a major role in protecting cells from prooxidants and electrophiles because it regulates basal and inducible expression of genes that contain antioxidant response element (ARE) sequences in their promoter regions. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). The ubiquitin ligase substrate adaptor kelch-like ECHassociated protein 1 (KEAP1) is a major repressor of NRF2. NRF2 is constantly degraded via the ubiquitin-proteasome pathway in a KEAP1-dependent manner. ROS or eletrophiles modify cysteine residues in KEAP1 causing loss of its adaptor activity, and in turn failure to ubiquitylate NRF2. Upon inactivation of KEAP1, NRF2 accumulates in the nucleus where it heterodimerizes with small Maf proteins and activates ARE-driven genes [4, 5]. Recent studies have shown that KEAP1-dependent ubiquitylation of NRF2 can be prevented by protein-protein interactions: these include the binding of p21 to NRF2 or the binding of p62/sequestosome to KEAP1 [6, 7]
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