Differential Yet Integral Contributions of Nrf1 and Nrf2 in the Human HepG2 Cells on Antioxidant Cytoprotective Response against Tert-Butylhydroquinone as a Pro-Oxidative Stressor

Abstract

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 caNrf2ΔN) 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 caNrf2ΔN 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.

Distinct Roles of Nrf1 and Nrf2 in Monitoring the Reductive Stress Response to Dithiothreitol (DTT)

“…Thereafter, these protein samples were collected and then subjected to measurement of their half-lives. In addition, trans -activity of Nrf1 and its mutants was determined by the pGL3-basic-6 × ARE-Luc reporter assay, as described previously [ 22 ].…”

“…However, the threshold of its generation or changes remains elusive to date, although it has been shown that the steady state of ER may be more easily disturbed [ 21 ]. Our previous work demonstrated differential and integral contributions of Nrf1 and Nrf2 to coordinately mediating distinct responsive gene expression profiles to the ER stressor tunicamycin (TU) [ 20 ] and pro-oxidative stressor tert -butylhydroquinone ( t BHQ) [ 22 ]. Recently, Nrf1, rather than Nrf2, was further identified as an indispensable redox-determining factor for mitochondrial homeostasis, in addition to the ER-associated proteostasis, by integrating multi-hierarchical responsive signaling pathways through nuclear respiratory gene controls towards mitochondrially located gene regulatory networks [ 23 ].…”

Nrf1 is an indispensable redox-determining factor for mitochondrial homeostasis by integrating multi-hierarchical regulatory networks

“…To further investigate distinct roles for Nrf1 and Nrf2 in the nuclear-to-mitochondrial regulation, the experimental Nrf1α –/– , Nrf2 –/– and WT cell lines were treated with tert -butylhydroquinone [ t BHQ, as a pro-oxidative stressor (48)]. The results revealed that Nrf1, Nrf2, PGC1α, αPal NRF1 , GABPα NRF2 , and TFAM (Figure 3C, D, S6A, B) along with HO-1 and GCLM (Figure S6C) were significantly induced by t BHQ stimulation in WT cells.…”

Distinct roles of Nrf1 and Nrf2 in monitoring the reductive stress response to dithiothreitol (DTT)

“…Thereafter, these protein samples were collected, and then subjected to measuring their half-lives. In addition, trans -activity of Nrf1 and its mutants was determined by the pGL3-basic-6 × ARE-Luc reporter assay as described previously (20).…”

“…Thereby, Nrf1 has been identified as an important ER sensor for changes in the intracellular redox, glucose, protein and lipid (including cholesterol) (18,19). Our previous work demonstrated differential and integral contributions of Nrf1 and Nrf2 to coordinately mediating distinct responsive gene expression profiles to the ER stressor tunicamycin (TU) (19) and pro-oxidative stressor tert -butylhydroquinone ( t BHQ)(20). Recently, Nrf1, rather than Nrf2, is further identified as an indispensable redox-determining factor for mitochondrial homeostasis, in addition to the ER-associated proteostasis, by integrating multi-hierarchical responsive signaling pathways through those nuclearly-located respiratory gene controls towards mitochondrially-located gene regulatory networks (21).…”