Modulation of FoxO signalling by glutathione

The highly conserved transcription factors of the Forkhead box O (FoxO) family are involved in the regulation of several cellular and physiological processes, including cell proliferation, apoptosis, oxidative stress response, longevity, and fuel metabolism. Here, in the context of the analysis of a redox modulation of FoxO proteins, the role of (a.) glutathione and (b.) FoxO1 cysteine residues in modulating FoxO activity were assessed employing cultured cells and an in vivo-model, Caenorhabditis elegans (C. elegans). Initially, the effects of diethyl maleate (DEM), an α,β-unsaturated carbonyl compound and thiol modulating agent, on C. elegans life span were explored. Low-dose-DEM elicited an extension of C. elegans life span. Using daf-16 or skn-1-deficient strains and through the upregulation of DAF-16 or SKN-1 target genes this life span extension was then demonstrated to be mediated by transcription factors DAF-16 and SKN-1, C. elegans homologues of FoxO and Nrf-2, respectively. In contrast, exposure to high-dose-DEM did not alter the expression of DAF-16 or SKN-1 targets and significantly shortened lifespan. Similarly, downregulation of glutathione biosynthesis through RNA interference to lower gcs-1 expression elicited the induced expression of genes encoding antioxidant proteins. In order to analyze the interaction between glutathione and a mammalian DAF-16 homolog, FoxO1, we investigated the role of FoxO1 cysteine residues in regulating FoxO1 activity under basal and stressful conditions. In cells transfected with plasmids encoding wildtype (WT) FoxO1 or cysteine-deficient FoxO1 forms, stimulation of FoxO1-responsive promoters was less prominent in cells overexpressing FoxO1-Cys1-7 as compared to those transfected with WT FoxO1. A single mutation in Cys612 mediated this loss of basal transactivation of FoxO1 activity, which in turn caused less prominent stimulation in expression of FoxO1 target genes as compared to overexpression of WT FoxO1. Exposure of cells to diamide, which promoted cellular S-glutathionylation, attenuated DNA binding of WT FoxO1, but not of FoxO1-Cys1-7. Diamide-induced S-glutathionylation was successfully reversed by overexpression of glutaredoxin (Grx)-1. In line with that, Grx-1 overexpression promoted an accelerated recovery of the impaired FoxO1 DNA binding. As only Grx-1 harboring an intact Cys23 was capable of accelerating recovery, whereas Cys26 was irrelevant, it was concluded that S-glutathionylation mediated diamide-induced attenuation of FoxO1 DNA binding. In summary, oxidative stress induced by thiol depletion regulates FoxO proteins, resulting in a non-linear effect of thiol depletion on C. elegans life span. Moreover, S-glutathionylation was identified as one mode of action of glutathione affecting FoxO1 activity under stressful conditions.


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