RXRα Inhibits the NRF2-ARE Signaling Pathway through a Direct Interaction with the Neh7 Domain of NRF2

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 (RXRa) is a hitherto unrecognized repressor of NRF2. RNAi-mediated knockdown of RXRa increased basal ARE-driven gene expression and induction of ARE-driven genes by the NRF2 activator tert-butylhydroquinone (tBHQ). Conversely, overexpression of RXRa decreased ARE-driven gene expression. Biochemical investigations showed that RXRa interacts physically with NRF2 in cancer cells and in murine small intestine and liver tissues. Furthermore, RXRa bound to ARE sequences in the promoters of NRF2-regulated genes. RXRa 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 RXRa 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, RXRa expression downregulated NRF2 and sensitized cells to the cytotoxic effects of therapeutic drugs. In summary, our findings show that RXRa diminishes cytoprotection by NRF2 by binding directly to the newly defined Neh7 domain in NRF2. Cancer Res; 73(10);

Emerging role of NRF2 in chemoresistance by regulating drug-metabolizing enzymes and efflux transporters

“…Neh6 has DSGIS and DSAPGS motifs that bind with b-transducin repeatcontaining protein (b-TrCP) which can negatively regulate NRF2 by recruiting S-phase kinase-associated protein 1 (Skp1)-Cul1-Rbx1 or Roc1 core E3 ubiquitin ligase (Chowdhry et al, 2013;Rada et al, 2011Rada et al, , 2012Wu et al, 2003). Neh7 domain can repress NRF2 by linking up with retinoid X receptor (Wang et al, 2013).…”

Butylated hydroxyanisole induces distinct expression patterns of Nrf2 and detoxification enzymes in the liver and small intestine of C57BL/6 mice

“…It has been found recently that glycogen synthase kinase-3 (GSK-3) catalyzes phosphorylation of the Neh6 domain in Nrf2 leading to ubiquitylation of the CNCbZIP protein by βTrCP (Chowdhry et al, 2013). Nuclear receptors also modulate Nrf2 activity in the nucleus (Wang et al, 2007(Wang et al, , 2013Namani et al, 2014). The role of Nrf2 in up-regulating the expression of phase II enzymes and other cytoprotective proteins has been demonstrated in vivo using Nrf2 knock-out mice (Itoh et al, 1997;McMahon et al, 2001).…”

Transcription factors Hsf1 and Nrf2 engage in crosstalk for cytoprotection

“…The newly synthesized Nrf2 accumulates, translocates to the nucleus, and binds (as a heterodimer with a small Maf protein) to antioxidant response elements (ARE, 5 0 -TGACnnnGC-3 0 ) in the upstream regulatory regions of its target genes [2][3][4]. In addition to Keap1, the activity of Nrf2 is also negatively regulated through glycogen synthase kinase (GSK)3/btransducin repeat-containing protein (b-TrCP)1-mediated degradation [17], or by interaction with retinoid X receptor (RXR)a [18].…”

“…( Figure IB) Nrf2 has seven functional domains, termed Nrf2-ECH homology (Neh)1-7 domains [11,18]. At the N terminus, Neh2 is the domain through which Nrf2 binds to its major negative regulator, Keap1: a dimeric substrate adaptor for the Rbx1 ubiquitin ligase complex.…”