Unification of Opposites between Two Antioxidant Transcription Factors Nrf1 and Nrf2 in Mediating Distinct Cellular Responses to the Endoplasmic Reticulum Stressor Tunicamycin

Abstract

The water-soluble Nrf2 (nuclear factor, erythroid 2-like 2, also called Nfe2l2) is accepted as a master regulator of antioxidant responses to cellular stress, and it was also identified as a direct target of the endoplasmic reticulum (ER)-anchored PERK (protein kinase RNA-like endoplasmic reticulum kinase). However, the membrane-bound Nrf1 (nuclear factor, erythroid 2-like 1, also called Nfe2l1) response to ER stress remains elusive. Herein, we report a unity of opposites between these two antioxidant transcription factors, Nrf1 and Nrf2, in coordinating distinct cellular responses to the ER stressor tunicamycin (TU). The TU-inducible transcription of <i>Nrf1</i> and <i>Nrf2</i>, as well as <i>GCLM</i> (glutamate cysteine ligase modifier subunit) and <i>HO-1</i> (heme oxygenase 1), was accompanied by activation of ER stress signaling networks. Notably, the unfolded protein response (UPR) mediated by ATF6 (activating transcription factor 6), IRE1 (inositol requiring enzyme 1) and PERK was significantly suppressed by Nrf1-specific knockout, but hyper-expression of Nrf2 and its target genes <i>GCLM</i> and <i>HO-1</i> has retained in<i>&nbsp;</i><i>Nrf1a^-/-</i> cells. By contrast,&nbsp;<i>Nrf2^-/-ΔTA</i> cells with genomic deletion of its transactivation (TA) domain resulted in significant decreases of GCLM, HO-1 and Nrf1; this was accompanied by partial decreases of IRE1 and ATF6, rather than PERK, but with an increase of ATF4 (activating transcription factor 4). Interestingly, Nrf1 glycosylation and its <i>trans</i>-activity to mediate the transcriptional expression of the 26S proteasomal subunits, were repressed by TU. This inhibitory effect was enhanced by&nbsp;<i>Nrf1a^-/-</i> and <i>Nrf2^-/-ΔTA</i><i>,</i> but not by a constitutive activator&nbsp;<i>caNrf2^ΔN</i> (that increased abundances of the non-glycosylated and processed Nrf1). Furthermore,&nbsp;<i>caNrf2^ΔN</i> also enhanced induction of PERK and IRE1 by TU, but reduced expression of ATF4 and HO-1. Thus, it is inferred that such distinct roles of Nrf1 and Nrf2 are unified to maintain cell homeostasis by a series of coordinated ER-to-nuclear signaling responses to TU. Nrf1 (i.e., a full-length form) acts in a cell-autonomous manner to determine the transcription of most of UPR-target genes, albeit Nrf2 is also partially involved in this process. Consistently, transactivation of ARE (antioxidant response element)-driven <i>BIP</i> (binding immunoglobulin protein)<i>-, PERK-</i> and <i>XBP1</i> (X-box binding protein 1)<i>-Luc</i> reporter genes was mediated directly by Nrf1 and/or Nrf2. Interestingly, Nrf1 is more potent than Nrf2 at mediating the cytoprotective responses against the cytotoxicity of TU alone plus tBHQ (<i>tert</i>-butylhydroquinone). This is also further supported by the evidence that the intracellular reactive oxygen species (ROS) levels are increased in&nbsp;<i>Nrf1a^-/-</i> cells, but rather are, to our surprise, decreased in<i>&nbsp;</i><i>Nrf2^-/-ΔTA</i> cells.