Keap1-dependent Proteasomal Degradation of Transcription Factor Nrf2 Contributes to the Negative Regulation of Antioxidant Response Element-driven Gene Expression

Abstract

Keap1 is a negative regulator of Nrf2, a bZIP transcription factor that mediates adaptation to oxidative stress. Previous studies suggested this negative regulation is a consequence of Keap1 controlling the subcellular distribution of Nrf2. We now report that Keap1 also controls the total cellular level of Nrf2 protein. In the RL34 non-transformed rat liver cell line, Nrf2 was found to accumulate rapidly in response to oxidative stress caused by treatment with sulforaphane, and the accumulation resulted from inhibition of proteasomal-mediated degradation of the bZIP protein. By heterologously expressing in COS1 cells epitope-tagged Nrf2 and an Nrf2 ⌬ETGE mutant lacking the Keap1-binding site, in both the presence and absence of Keap1 we demonstrate that Nrf2 is subject to ubiquitination and proteasomal degradation independently of both Keap1 and the redox environment of the cell. In oxidatively stressed cells, this is the sole mechanism responsible for Nrf2 degradation. However, under homeostatic conditions Nrf2 is subject to a substantially more rapid mode of proteasomal degradation than it is in oxidatively stressed cells, and this rapid turnover of Nrf2 requires it to interact with Keap1. Within Nrf2, the N-terminal Neh2 domain is identified as the redox-sensitive degron. These data suggest that Keap1 negatively regulates Nrf2 by both enhancing its rate of proteasomal degradation and altering its subcellular distribution.

Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis

“…However, in response to oxidative stress, modification of reactive cysteines within Keap1 protein induces a conformational change leading to Nrf2 stabilization with increased expression of Nrf2-downstream target detoxifying and antioxidant genes. 17 Thus we observe in our study that Keap1 deficiency in liver cells induces Nrf2 protein expression and function and increases expression of antioxidant enzyme GCLc. However, despite augmenting Nrf2 function, Keap1 silencing induced spontaneous cell death, suggesting that the beneficial increase in antioxidant levels induced by loss of Keap1 is overridden by simultaneous activation of pro-death signals (e.g., Bim and PUMA).…”

“…16 Kelch-like ECH-associated protein (Keap)-1 is a bric-à -brac/Kelch protein that functions as an adaptor protein for Cul3-based E3 ligase and initiates the proteasomal degradation of various proteins, such as nuclear factor (erythroidderived 2)-like 2 (Nrf2), a transcription factor that regulates the expression of various antioxidant enzymes. 17 Keap1 null mice have a lethal phenotype, 18 but mice bearing an hepatocyte specific disruption of the Keap1 gene (Keap1(flox/-)::Albumin-Cre) are viable and display high levels of Nrf2 protein in their liver. 19 Although genetic deficiency in Keap1 rendered mice more resistant to APAPinduced hepatotoxicity, loss of Keap1 compromised the longterm survival of these animals without increasing their risk of tumor formation.…”

Activation of the Nrf2/antioxidant response pathway increases IL-8 expression

“…Finally, to demonstrate that activation of endogenous Nrf2 also increases IL-8 production, cells were treated with the proteasome inhibitor MG-132, which activates the Nrf2/antioxidant pathway by preventing Nrf2 degradation [17][18][19]. IL-8 production was significantly increased in mesangial cells treated with MG-132, and this was completely blocked by prior infection of the cells with Ad.DN-Nrf2 (Fig.…”

Transcription factor NFE2L2/NRF2 modulates chaperone-mediated autophagy through the regulation of LAMP2A

“…HEK293T cells were grown on 10-cm plates until they reached 85% confluence and transfected with an NFE2L2 expression plasmid that lacks the high affinity binding site for KEAP1 and contains a V5 tag (NFE2L2 ΔETGE -V5) [51]. Briefly, cells were cross-linked with 1% formaldehyde (Fluka, 47,630) and the reaction was stopped with 125 mM glycine (Bio-Rad, 161–0718).…”