Nrf2 is an inhibitor of the Fas pathway as identified by Achilles' Heel Method, a new function-based approach to gene identification in human cells

Abstract

Here we describe the Achilles' Heel Method (AHM), a new function-based approach for identification of inhibitors of signaling pathways, optimized for human cells. The principle of AHM is the identification of 'sensitizing' cDNAs based on their decreased abundance following selection. As a proof of principle, we have employed AHM for the identification of Fas/CD95/APO-1 pathway inhibitors. HeLa cells were transfected with an antisense cDNA expression library in an episomal vector followed by selection with a suboptimal dose of the apoptotic inducer. Antisense inactivation of Fas inhibitors rendered the cells more sensitive to apoptosis resulting in their preferential death and consequent loss of their sensitizing episomes that were identified by subtraction. We show that the resulting products were enriched for sensitizing cDNAs as seven out of eight candidates tested were confirmed as inhibitors of Fas-induced killing either by transfection or by pharmacological inhibition. Furthermore, we demonstrate by multiple approaches that one candidate, NF-E2 related factor 2 (Nrf2), is an inhibitor of Fas-induced apoptosis. Inactivation of Nrf2 by antisense or by a membrane permeable dominantnegative polypeptide sensitized cells while overexpression of Nrf2 protected cells from Fas-induced apoptosis. In addition, dicumarol, an inhibitor of the phase II detoxifying enzyme NQO1, a downstream target of Nrf2, sensitized cells. Nrf2 induces the production of Glutathione (GSH) and we demonstrated that N-acetyl L-cysteine (NAC), a precursor to GSH, protected cells from Fas-mediated killing. Taken together, AHM is a powerful approach for the identification of inhibitors of a signaling pathway with a low rate of false positives that opens new avenues for function profiling of human genes and discovery of new drug targets.

Covalent Modification at Cys151 Dissociates the Electrophile Sensor Keap1 from the Ubiquitin Ligase CUL3

“…Nrf2 exhibits a proclivity for dimerization with the transcription factors c-Jun, Jun B, Jun D, ATF3, ATF4, and small Mafs (3)(4)(5)(6). Heterodimeric binding to antioxidant response elements (ARE: 5′-(A/G)TGA (C/G) NNNGC-3′) (7,8) located in the proximal promoter of target genes initiates Nrf2-directed expression of more than 200 gene products, such as those involved in phase II detoxication, antioxidant gene expression, apoptosis, cell growth, and proteasomal activity (9) (2,10). Nrf2 regulates both the constitutive and inducible expression of this stress response network and may be considered a node for the integration of stress signaling.…”

Nrf2 regulates the sensitivity of death receptor signals by affecting intracellular glutathione levels

“…The reports strongly indicated that Nrf2 activity may influence the sensitivity of death signaling, but the relationship between Nrf2 and apoptotic signaling was not clearly characterized. Recently, Kotlo et al (2003) demonstrated that inactivation of Nrf2 by antisense or by using a membranepermeable dominant-negative polypeptide sensitized cells while, conversely, overexpression of Nrf2 protected cells from Fas-induced apoptosis. In addition, they demonstrated that N-acetyl-l-cysteine (NAC), a precursor to GSH, protected cells from Fas-mediated killing in HeLa cells.…”

Protection from mitochondrial complex II inhibitionin vitroandin vivoby Nrf2-mediated transcription

“…Consequently, Nrf2-induced ARE activation coordinates the expression of many genes involved in combating oxidative stress and toxicity in a wide variety of tissues and cell types (35)(36)(37)(38)(39)(40)(41). In addition to protecting against chemical insults, carcinogenesis, and aging (41)(42)(43)(44), Nrf2 has been shown to directly inhibit Fas-mediated apoptosis, a substrate for caspase-3-like proteases and an effector of PK-like ER kinase-mediated cell survival (45)(46)(47)(48).…”