Regulation of Nrf2 by Mitochondrial Reactive Oxygen Species in Physiology and Pathology

Abstract

Reactive oxygen species (ROS) are byproducts of aerobic respiration and signaling molecules that control various cellular functions. Nrf2 governs the gene expression of endogenous antioxidant synthesis and ROS-eliminating enzymes in response to various electrophilic compounds that inactivate the negative regulator Keap1. Accumulating evidence has shown that mitochondrial ROS (mtROS) activate Nrf2, often mediated by certain protein kinases, and induce the expression of antioxidant genes and genes involved in mitochondrial quality/quantity control. Mild physiological stress, such as caloric restriction and exercise, elicits beneficial effects through a process known as "mitohormesis." Exercise induces NOX4 expression in the heart, which activates Nrf2 and increases endurance capacity. Mice transiently depleted of SOD2 or overexpressing skeletal muscle-specific UCP1 exhibit Nrf2-mediated antioxidant gene expression and PGC1α-mediated mitochondrial biogenesis. ATF4 activation may induce a transcriptional program that enhances NADPH synthesis in the mitochondria and might cooperate with the Nrf2 antioxidant system. In response to severe oxidative stress, Nrf2 induces Klf9 expression, which represses mtROS-eliminating enzymes to enhance cell death. Nrf2 is inactivated in certain pathological conditions, such as diabetes, but Keap1 down-regulation or mtROS elimination rescues Nrf2 expression and improves the pathology. These reports aid us in understanding the roles of Nrf2 in pathophysiological alterations involving mtROS.

Keywords: ATF4; Keap1; Klf9; Nrf2; PGC1α; Sirt6; mitochondrial ROS.

Figure 1

Mitochondrial ROS (mtROS) production and Nrf2 activation by temporal SOD2 depletion, bacterial infection, and Down syndrome. Superoxide (O₂ ^{∙ −}) produced in the mitochondrial matrix is readily reduced by SOD2 to hydrogen peroxide (H₂O₂), which is then reduced by glutathione peroxidase (GPX) to water [12]. Short-term SOD2 depletion in mouse embryos increases mtROS; however, at the postnatal stage, it activates antioxidant defense by Nrf2 and remodels mitochondrial function in the liver [13]. In fibroblasts derived from Down syndrome patients, elevated mtROS production is counteracted by Nrf2, which is activated by PKCδ-mediated phosphorylation [14]. The expression of mitochondrially targeted catalase (mtCAT) alleviates oxidative stress and inhibits Nrf2 activation in fibroblasts from Down syndrome patients [14]. Bacterial infection stimulates macrophages to induce ROS production via NADPH oxidase, and mitochondria are used to eliminate phagocytosed bacteria [15]. Mst1/Mst2 regulates the recruitment of mitochondria to phagosomes and activates Nrf2 by phosphorylating Keap1 to protect the host from excess ROS [15]. Nrf2 induces the expression of cytoplasmic enzymes as well as genes involved in mitochondrial biogenesis and quality control, such as NRF-1, Pink1 and SOD2 [16,17,18,19].

Figure 2

Mitohormesis caused by mild metabolic stress elicits an antioxidant defense response. Metabolic stress, such as caloric restriction and exercise, causes a decline in ATP levels and an increase in the NAD⁺/NADH ratio that is sensed by downstream pathways that promote mitochondrial function and increase ATP synthesis [50,55]. An increase in AMP/ATP activates AMPK, which induces mitochondrial biogenesis via PGC1α activation and enhances autophagy via mTOR inhibition. An increase in NAD⁺/NADH activates the NAD⁺-dependent deacetylases SIRT1 and SIRT3 [61]. Mitochondrially localized SIRT3 activates enzymes involved in mitochondrial ATP synthesis, whereas nuclear SIRT1 also activates PGC1α [50,55]. PGC1α activates the expression of genes involved in mitochondrial biogenesis, fatty acid oxidation, oxidative phosphorylation (OxPhos) and the antioxidant defense system. These adaptations resolve energy shortages and improve metabolic health, exercise capacity and longevity [3].

Figure 3

Acute exercise activates Nrf2 via NOX4 expression in the heart. Acute exercise stress (AES) stimulates cardiac performance and exercise capacity via the expression of genes involved in mitochondrial quality control (PRDX3, TXNRD2, and SOD2) as well as cytoplasmic ROS scavenging (GCLC, GSTA1/2) in a NOX4- and Nrf2-dependent manner [73,75]. NOX4 induced by AES localizes to various organelles, including the plasma membrane, nucleus, endoplasmic reticulum (ER), and especially mitochondria, in the heart and produces ROS to activate Nrf2 [78,79,80]. The AES-induced decrease in mtROS is inhibited by Nrf2 deficiency but rescued by the mitochondria-targeted antioxidant MitoQ [73].

Figure 4

Weak and strong oxidative stresses induce differential gene expression via the Nrf2 and Klf9 axes. Nrf2 induces the expression of cytoprotective genes, such as HO-1 and NQO1, in response to a wide range of oxidative stresses [88]. High-dose H₂O₂ or sulforaphane induces Klf9 expression, which represses Txnrd2 gene expression as well as that of other Nrf2-target genes, such as Prdx6, and induces cytotoxic ROS production [88,89]. The line graphs represent the oxidative stress level (x-axis) and transcription (y-axis) relationship.

Figure 5

Regulation of gene expression by Nrf2 and Sirt6 crosstalk. Sirt6 expression restores Nrf2 and Sirt3 expression and improves diabetic cardiomyopathy. Sirt6 activates Nrf2 by repressing Keap1 expression as well as interfering with the Nrf2-Keap1 interaction [100]. Sirt6 expression is also important for hMSC maintenance and acts by enhancing Nrf2 target gene expression via epigenetic modifications, increased histone H3 Lys4 trimethylation and decreased Lys56 acetylation [98]. Glutamine starvation in autophagy-deficient cells induces the expression of amino acid transporters (ATTs) to replenish cellular amino acid shortages [106]. Nrf2 is recruited to the ATT gene enhancer by ATF4, where the replacement of Sirt6 with Nrf2 enhances H3K56 acetylation and AAT transcription [106].

Figure 6

Cooperative regulation of genes involved in glutathione (GSH) and NADPH synthesis by Nrf2 and ATF4. Carnosic acid activates both Nrf2 and ATF4 and induces antioxidant genes and genes involved in amino acid synthesis/transport and GSH synthesis. The contributions of Nrf2 and ATF4 to the gene activation were analyzed by knockdown of Nrf2 or ATF4 and shown with color characters ([124] and our unpublished observation). Induction of cystine transporter xCT and GSH synthetic enzymes (GCLC, GCLM, GSS, and GSR) was dependent on both Nrf2 and ATF4 (genes indicated with blue). Genes involved in serine synthesis (PHGDH, PSAT1, and PSPH), transsulfuration (CBS and CTH), folate cycle (SHMT2 and MTHFD2), and asparagine synthase (ASNS) and Ala/Ser/Cys/Thr transporter-1 and -2 (ASCT1/2) are dependent on only ATF4 and indicated with red. Colored genes are also up-regulated in HSA-UCP1-Tg mice [132] except for ASCT1 and additionally include G6PD, PEPCK-C, PEPCK-M and MTHFD1 (indicted with black). G6PD is a rate-limiting enzyme of pentose phosphate pathway (PPP) that supplies NADPH and is known as an Nrf2 target gene [28]. PEPCKs may redirect TCA cycle metabolites to Ser synthetic pathway and are reported as ATF4-regulated genes [135,136]. Folate cycle converts Ser to Gly and up-regulation of mitochondrial SHMT2 and MTHFD2 may increase mitochondrial NADPH [29].