Nuclear trapping of inactive FOXO1 by the Nrf2 activator diethyl maleate

Andrea Gille,Abdullah Turkistani,Dimitrios Tsitsipatis,Xiaoqing Hou,Sarah Tauber,Ingrit Hamann,Nadine Urban,Katrin Erler,Holger Steinbrenner,Lars-Oliver Klotz

doi:10.1016/j.redox.2018.09.010

Andrea Gille, Abdullah Turkistani + Show 8 more

Open Access

https://doi.org/10.1016/j.redox.2018.09.010

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Abstract

Diethyl maleate (DEM), a thiol-reactive α,β-unsaturated carbonyl compound, depletes glutathione (GSH) in exposed cells and was previously shown by us to elicit a stress response in Caenorhabditis elegans that, at lower concentrations, results in enhanced stress resistance and longer lifespan. This hormetic response was mediated through both the Nrf2 ortholog, SKN-1, and the forkhead box O (FOXO) family transcription factor DAF-16. As FOXO signaling is evolutionarily conserved, we analyzed here the effects of DEM exposure on FOXO in cultured human cells (HepG2, HEK293). DEM elicited nuclear accumulation of GFP-coupled wild-type human FOXO1, as well as of a cysteine-deficient FOXO1 mutant. Despite the nuclear accumulation of FOXO1, neither FOXO1 DNA binding nor FOXO target gene expression were stimulated, suggesting that DEM causes nuclear accumulation but not activation of FOXO1. FOXO1 nuclear exclusion elicited by insulin or xenobiotics such as arsenite or copper ions was attenuated by DEM, suggesting that DEM interfered with nuclear export. In addition, insulin-induced FOXO1 phosphorylation at Thr-24, which is associated with FOXO1 nuclear exclusion, was attenuated upon exposure to DEM. Different from FOXO-dependent expression of genes, Nrf2 target gene mRNAs were elevated upon exposure to DEM. These data suggest that, different from C. elegans, DEM elicits opposing effects on the two stress-responsive transcription factors, Nrf2 and FOXO1, in cultured human cells.

Highlights

The intracellular tripeptide thiol, glutathione, is generally known as a crucial contributor to cellular antioxidant defense systems as well as to xenobiotic metabolism [1]
We analyzed FOXO1 subcellular localization upon exposure to Diethyl maleate (DEM) in two different types of cultured human cells, HepG2 cells and HEK293 human embryonic kidney cells, using GFP-FOXO1 constructs. We used these two cell types because of their different patterns in basal subcellular FOXO1 distribution that correlates with basal Akt activity and FOXO1 phosphorylation levels as previously described [12]: Whereas HEK293 cells have a higher fraction of cells with predominantly cytoplasmic FOXO1 under basal growth conditions than HepG2 cells, the latter have a higher percentage of cells in an intermediate state with respect to FOXO1 distribution, as seen in Fig. 2C
We investigated the effects of a known Nrf2 activator, diethyl maleate (DEM), on forkhead box O (FOXO) transcription factors in mammalian cells: We demonstrate that exposure of mammalian cells to DEM causes nuclear accumulation of FOXO1 transcription factor, but no stimulation of FOXO-dependent gene expression

Summary

Introduction

The intracellular tripeptide thiol, glutathione (γGlu-Cys-Gly; GSH), is generally known as a crucial contributor to cellular antioxidant defense systems as well as to xenobiotic metabolism [1]. It was previously tested for its role in the regulation of stress resistance and life span in the model organism, Caenorhabditis elegans. Diethyl maleate (DEM), an α,β-unsaturated carbonyl compound that is frequently used for the depletion of cellular GSH, and a known stimulator of Nrf signaling in mammalian cells [3,4,5], elicited an increase in stress resistance in C. elegans [2]. SKN-1, the ortholog of Nrf, and DAF-16, the C. elegans ortholog of mammalian

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