Mechanism of chemical activation of Nrf2 - PubMed (original) (raw)

Mechanism of chemical activation of Nrf2

Yun Li et al. PLoS One. 2012.

Abstract

NF-E2 related factor-2 (Nrf2) promotes the transcription of many cytoprotective genes and is a major drug target for prevention of cancer and other diseases. Indeed, the cancer-preventive activities of several well-known chemical agents were shown to depend on Nrf2 activation. It is well known that chemopreventive Nrf2 activators stabilize Nrf2 by blocking its ubiquitination, but previous studies have indicated that this process occurs exclusively in the cytoplasm. Kelch-like ECH-associated protein 1 (Keap1) binds to Nrf2 and orchestrates Nrf2 ubiquitination, and it has been a widely-held view that inhibition of Nrf2 ubiquitination by chemopreventive agents results from the dissociation of Nrf2 from its repressor Keap1. Here, we show that while the activation of Nrf2 by prototypical chemical activators, including 5,6-dihydrocyclopenta-1,2-dithiole-3-thione (CPDT) and sulforaphane (SF), results solely from inhibition of its ubiquitination, such inhibition occurs predominantly in the nucleus. Moreover, the Nrf2 activators promote Nrf2 association with Keap1, rather than disassociation, which appears to result from inhibition of Nrf2 phosphorylation at Ser40. Available evidence suggests the Nrf2 activators may block Nrf2 ubiquitination by altering Keap1 conformation via reaction with the thiols of specific Keap1 cysteines. We further show that while the inhibitory effects of CPDT and SF on Nrf2 ubiquitination depend entirely on Keap1, Nrf2 is also degraded by a Keap1-independent mechanism. These findings provide significant new insight about Nrf2 activation and suggest that exogenous chemical activators of Nrf2 enter the nucleus to exert most of their inhibitory impact on Nrf2 ubiquitination and degradation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. CPDT and SF stimulate Nrf2 transactivation activity by stabilizing its protein.

(A) Chemical structure of CPDT and SF. RT-4 cells and NBT-II cells were treated with CPDT (50 µM), SF (8 µM) or vehicle (0.1% DMSO) for 6 or 24 h. (B) Whole cell lysates were then prepared for IB or (C) total RNA was isolated for RT-PCR analysis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control.

Figure 2

Figure 2. CPDT and SF block only Keap1-mediated Nrf2 degradation and require key cysteine residues of Keap1.

(A) Murine embryonic fibroblasts (MEF), including wild type MEF (Keap1+/+) and MEF with Keap1 knockout (Keap1−/−), were treated with vehicle, MG132 (25 µM), CPDT (50 µM) or SF (8 µM) for 6 h. Cells were then harvested for IB of Nrf2 and GAPDH. (B) RT-4 cells were transfected with either a control siRNA or a specific Keap1-targeting siRNA for 48 h, followed by treatment with vehicle, MG132 (25 µM), CPDT (50 µM) or SF (8 µM) for 6 h. Cells were then harvested for IB of Nrf2, Keap1 and GAPDH. (C) MEF with knockout of both Keap1 and Nrf2 were mock transfected or transfected with expression vectors of Nrf2 (pEF/Nrf2) with or without Keap1 (wild type or one of three Keap1 mutants, all cloned to pcDNA3) for 48 h, followed by treatment with vehicle, CPDT (50 µM) or SF (8 µM) for 6 h. Whole cell lysates were then prepared for IB.

Figure 3

Figure 3. CPDT and SF block Nrf2 degradation mainly in the nucleus, but do not dissociate the Nrf2-Keap1 complex or the ubiquitination complex.

RT-4 cells and NBT-II cells were treated with vehicle, CPDT (50 µM) or SF (8 µM) for 6 h, from which cytosols and nuclear extracts were prepared and were subjected to analysis by IB or IP followed by IB. The loading amount of the nuclear sample was about half of the cytoplasmic sample for both IP and IB. (A) IB of indicated proteins. GAPDH and lamin A were used to confirm the purity of the cytosols and nuclear extracts, respectively. (B) Cytosols and nuclear extracts were subjected to IP with anti-Keap1, followed by IB of the indicated proteins.

Figure 4

Figure 4. CPDT and SF block Nrf2 ubiquitination.

NBT-II cells were co-transfected for 24 h with expression plasmids for Nrf2 (pEF/Nrf2), Keap1 (pcDNA1/Keap1) or ubiquitin (pMT107-His-Ub, a polyhistidine-tagged ubiquitin expression plasmid), followed by treatment for 6 h with MG132 (25 µM), MG132 (25 µM) plus CPDT (50 µM), or MG132 (25 µM) plus SF (8 µM), from which cytoplasmic and nuclear samples were prepared and analyzed by IB of various proteins. For detection of ubiquitinated Nrf2, the samples were prepared under denatured conditions and then subjected to IP with anti-Nrf2, followed by IB with an anti-His-HRP-conjugated antibody (for detection of ubiquitinated Nrf2). Equal amounts of nuclear and cytoplasmic proteins were used. The arrow points to the Ub band.

Figure 5

Figure 5. The paradigm of chemical activation of Nrf2.

The Keap1-mediated Nrf2 ubiquitination and proteasomal degradation exist in both cytoplasm and nucleus. Nrf2 activators block Nrf2 ubiquitination by causing conformational change of Keap1 through reaction with its critical cysteine residues (C273 and C288), and this process occurs primarily in the nucleus. Keap1 is shown as a monomer in this model, but a previous study suggests that Nrf2 may be associated with Keap1 homodimer .

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