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Nrf2 is activated by disruption of mitochondrial thiol homeostasis but not by enhanced mitochondrial superoxide production.

ABSTRACT: The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates the expression of genes involved in antioxidant defenses to modulate fundamental cellular processes such as mitochondrial function and GSH metabolism. Previous reports proposed that mitochondrial reactive oxygen species production and disruption of the GSH pool activate the Nrf2 pathway, suggesting that Nrf2 senses mitochondrial redox signals and/or oxidative damage and signals to the nucleus to respond appropriately. However, until now, it has not been possible to disentangle the overlapping effects of mitochondrial superoxide/hydrogen peroxide production as a redox signal from changes to mitochondrial thiol homeostasis on Nrf2. Recently, we developed mitochondria-targeted reagents that can independently induce mitochondrial superoxide and hydrogen peroxide production mitoParaquat (MitoPQ) or selectively disrupt mitochondrial thiol homeostasis MitoChlorodinitrobenzoic acid (MitoCDNB). Using these reagents, here we have determined how enhanced generation of mitochondrial superoxide and hydrogen peroxide or disruption of mitochondrial thiol homeostasis affects activation of the Nrf2 system in cells, which was assessed by the Nrf2 protein level, nuclear translocation, and expression of its target genes. We found that selective disruption of the mitochondrial GSH pool and inhibition of its thioredoxin system by MitoCDNB led to Nrf2 activation, whereas using MitoPQ to enhance the production of mitochondrial superoxide and hydrogen peroxide alone did not. We further showed that Nrf2 activation by MitoCDNB requires cysteine sensors of Kelch-like ECH-associated protein 1 (Keap1). These findings provide important information on how disruption to mitochondrial redox homeostasis is sensed in the cytoplasm and signaled to the nucleus.

SUBMITTER: Cvetko F

PROVIDER: S-EPMC7948991 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Superoxide radical plays an important role in redox cell signaling and physiological processes; however, overproduction of superoxide or insufficient activity of antioxidants leads to oxidative stress and contributes to the development of pathological conditions such as endothelial dysfunction and hypertension. Meanwhile, the studies of superoxide in biological systems represent unique challenges associated with short lifetime of superoxide, insufficient reactivity of the superoxide probes, and lack of site-specific detection of superoxide. In this work we have developed 15N-and deuterium-enriched spin probe 15N-CAT1H for high sensitivity and site-specific detection of extracellular superoxide. We have tested simultaneous tracking of extracellular superoxide by 15N-CAT1H and intramitochondrial superoxide by conventional 14N-containing spin probe mitoTEMPO-H in immune cells isolated from spleen, splenocytes, under basal conditions or stimulated with inflammatory cytokines IL-17A and TNFα, NADPH oxidase activator PMA, or treated with inhibitors of mitochondrial complex I rotenone or complex III antimycin A. 15N-CAT1H provides two-fold increase in sensitivity and improves detection since EPR spectrum of 15N-CAT1 nitroxide does not overlap with biological radicals. Furthermore, concurrent use of cell impermeable 15N-CAT1H and mitochondria-targeted 14N-mitoTEMPO-H allows simultaneous detection of extracellular and mitochondrial superoxide. Analysis of IL-17A- and TNFα-induced superoxide showed parallel increase in 15N-CAT1 and 14N-mitoTEMPO signals suggesting coupling between phagocytic NADPH oxidase and mitochondria. The interplay between mitochondrial superoxide production and activity of phagocytic NADPH oxidase was further investigated in splenocytes isolated from Sham and angiotensin II infused C57Bl/6J and Nox2KO mice. Angiotensin II infusion in wild-type mice increased the extracellular basal splenocyte superoxide which was further enhanced by complex III inhibitor antimycin A, mitochondrial uncoupling agent CCCP and NADPH oxidase activator PMA. Nox2 depletion attenuated angiotensin II mediated stimulation and inhibited both extracellular and mitochondrial PMA-induced superoxide production. These data indicate that splenocytes isolated from hypertensive angiotensin II-infused mice are "primed" for enhanced superoxide production from both phagocytic NADPH oxidase and mitochondria. Our data demonstrate that novel 15N-CAT1H provides high sensitivity superoxide measurements and combination with mitoTEMPO-H allows independent and simultaneous detection of extracellular and mitochondrial superoxide. We suggest that this new approach can be used to study the site-specific superoxide production and analysis of important sources of oxidative stress in cardiovascular conditions.

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Nrf2 is activated by disruption of mitochondrial thiol homeostasis but not by enhanced mitochondrial superoxide production.

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