In the past 25 years, Nrf2 (nuclear factor erythroid 2-related factor 2, also called NFE2L2) had been preferentially parsed as a master hub of regulating antioxidant, detoxification, and cytoprotective genes; albeit as a matter of fact that Nrf1 (nuclear factor erythroid 2-related factor 1, also called NFE2L1)—rather than Nrf2—is indispensable for cell homeostasis and organ integrity during normal growth and development. Herein, distinct genotypic cell lines (i.e., Nrf1α^−/−, Nrf2^−/−ΔTA, and caNrf2^ΔN) are employed to determine differential yet integral roles of Nrf1 and Nrf2 in mediating antioxidant responsive genes to tert-butylhydroquinone (tBHQ) serving as a pro-oxidative stressor. In Nrf1α^−/− cells, Nrf2 was highly accumulated but also could not fully compensate specific loss of Nrf1α’s function in its basal cytoprotective response against endogenous oxidative stress, though it exerted partially inducible antioxidant response, as the hormetic effect of tBHQ, against apoptotic damages. By contrast, Nrf2^−/−ΔTA cells gave rise to a substantial reduction of Nrf1 in both basal and tBHQ-stimulated expression levels and hence resulted in obvious oxidative stress, but it can still be allowed to mediate a potent antioxidant response, as accompanied by a significantly decreased ratio of GSSG (oxidized glutathione) to GSH (reduced glutathione). Conversely, a remarkable increase of Nrf1 expression resulted from the constitutive active caNrf2^ΔN cells, which were not manifested with oxidative stress, whether or not it was intervened with tBHQ. Such inter-regulatory effects of Nrf1 and Nrf2 on the antioxidant and detoxification genes (encoding HO-1, NQO1, GCLC, GCLM, GSR, GPX1, TALDO, MT1E, and MT2), as well on the ROS (reactive oxygen species)-scavenging activities of SOD (superoxide dismutase) and CAT (catalase), were further investigated. The collective results unraveled that Nrf1 and Nrf2 make distinctive yet cooperative contributions to finely tuning basal constitutive and/or tBHQ-inducible expression levels of antioxidant cytoprotective genes in the inter-regulatory networks. Overall, Nrf1 acts as a brake control for Nrf2’s functionality to be confined within a certain extent, whilst its transcription is regulated by Nrf2.

Transcription factor Nrf2 (nuclear factor, erythroid 2-like 2, encoded by Nfe2l2) has been accepted as a key player in redox regulatory responses to oxidative or reductive stresses. However, relatively little is known about the potential role of Nrf1 (nuclear factor, erythroid 2-like 1, encoded by Nfe2l1) in the redox responses, particularly to reductive stress, although this ‘fossil-like’ factor is indispensable for cell homeostasis and organ integrity during the life process. Herein, we examine distinct roles of Nrf1 and Nrf2 in monitoring the defense response to 1,4–dithiothreitol (DTT, serving as a reductive stressor), concomitantly with unfolded protein response being induced by this chemical (also defined as an endoplasmic reticulum stressor). The results revealed that intracellular reactive oxygen species (ROS) were modestly increased in DTT-treated wild-type (WT) and Nrf1α^−/− cell lines, but almost unaltered in Nrf2^−/−ΔTA or caNrf2^ΔN cell lines (with a genetic loss of transactivation or N-terminal Keap1-binding domains, respectively). This chemical treatment also enabled the rate of oxidized to reduced glutathione (i.e., GSSG to GSH) to be amplified in WT and Nrf2^−/−ΔTA cells, but diminished in Nrf1α^−/− cells, along with no changes in caNrf2^ΔN cells. Consequently, Nrf1α^−/−, but not Nrf2^−/−ΔTA or caNrf2^ΔN, cell viability was reinforced by DTT against its cytotoxicity, as accompanied by decreased apoptosis. Further experiments unraveled that Nrf1 and Nrf2 differentially, and also synergistically, regulated DTT-inducible expression of critical genes for defending against redox stress and endoplasmic reticulum stress. In addition, we also identified that Cys342 and Cys640 of Nrf1 (as redox-sensing sites within its N-glycodomain and DNA-binding domain, respectively) are required for its protein stability and transcription activity.

Nrf2 (nuclear factor E2-related factor 2, encoded by Nfe2l2) acts as a master transcriptional regulator in mediating antioxidant, detoxification, and cytoprotective responses against oxidative, electrophilic, and metabolic stress, but also plays a crucial role in cancer metabolism and multiple oncogenic pathways, whereas the redox sensor Keap1 functions as a predominant inhibitor of Nrf2 and, hence, changes in its expression abundance directly affect the Nrf2 stability and transcriptional activity. However, nuanced functional isoforms of Keap1 α and β have rarely been identified to date. Herein, we have established four distinct cell models stably expressing Keap1^−/−, Keap1β(Keap1^Δ1–31), Keap1-Restored, and Keap1α-Restored aiming to gain a better understanding of similarities and differences of two Keap1 isoforms between their distinct regulatory profiles. Our experimental evidence revealed that although Keap1 and its isoforms are still localized in the cytoplasmic compartments, they elicited differential inhibitory effects on Nrf2 and its target HO-1. Furthermore, transcriptome sequencing unraveled that they possess similar but different functions. Such functions were further determined by multiple experiments in vivo (i.e., subcutaneous tumour formation in nude mice) and in vitro (e.g., cell cloning, infection, migration, wound healing, cell cycle, apoptosis, CAT enzymatic activity, and intracellular GSH levels). Of note, the results obtained from tumourigenesis experiments in xenograft model mice were verified based on the prominent changes in the PTEN signaling to the PI3K-AKT-mTOR pathways, in addition to substantially aberrant expression patterns of those typical genes involved in the EMT (epithelial–mesenchymal transition), cell cycle, and apoptosis.

Ion: Curcumin (CUR), a naturally occurring phenolic small molecule, has been extensively applied in the treatment of diverse diseases for over half a century. Currently, this chemical has also been verified to exhibit a wide array of its biopharmacological activities, such as enhancing immunity, possessing antiviral, anti-cardiovascular, and anti-cancer properties. Besides, also as a nutritional supplement, CUR enables inflammatory prevention and enhance the combined efficacy of it with chemotherapy. Such characteristic of safeguarding normal organisms while treating or alleviating diseases is manifested as a common bidirectional regulation and its hormetin effect of phenolic compounds, particularly in response to stress. In this redox process, two antioxidant transcription factors Nrf1 and Nrf2 (encoded by Nfe2l1 and Nfe2l2 , respectively) play distinct and crucial roles. Thereby, we investigated their expression profiles of genes regulated by Nrf1 and Nrf2 in different signaling responses modulated by CUR. The resulting evidence has been provided, demonstrating that distinctive cellular metabolisms, molecular pathways, and signaling mechanisms account for Nrf1 and Nrf2, as drug targets. Both factors also play diverse roles in the anticancer effects of CUR on HepG2 cells and xenograft mice. However, the effect of CUR on xenograft tumors in vivo is not entirely satisfactory, although such anti-cancer effect was achieved by promoting Nrf1 expression, it appeared more reliant on Nrf2 (particularly in the absence of Nrf1). As such, we unexpectedly discovered there is not a simple regulatory relationship between CUR and Nrf2. This is supported by substantial inhibition of Nrf2 by CUR in the aberrantly proliferating Nrf1α ^–/– cells, even albeit this chemical stimulates the expression of Nrf1 and Nrf2 in wild-type cells.