Role of Nrf2 stress response pathway in physiology and diseases
Oxidative stress has been implicated in the pathogenesis of many age-related diseases such as Alzheimer's disease, age-related macular degeneration, and diabetes. Goal of this project is to develop a strategy for preventing age-related diseases targeting Nrf2-mediated cytoprotective mechanisms. Nrf2 is transcription factor that forms heterodimer with small Maf proteins to bind its target sequence antioxidant responsive element (ARE) (1, 2). Normally, Nrf2 is trapped to the E3 ligase adaptor Kelch-like ECH-associated protein 1 (Keap1) protein and subsequently degraded by the proteasome. However, when reactive cysteine residues of Keap1 protein are modified by oxidative stress or electrophilic substances, degradation of Nrf2 is inhibited, and accumulated Nrf2 translocates to the nucleus to bind to the ARE.
A. Elucidation of Nrf2 transcriptional network To elucidate the transcriptional network mediated by Nrf2, we previously performed yeast two hybrid screening using the C-terminus of Nrf2 that contains basic leucine zipper region of Nrf2. Interestingly, most positive clones encoded ATF4 (unpublished data, see also ref 3). Nrf2 and ATF4 are activated by the proteasome inhibition, cooperatively activates xCT expression which increases the intracellular cysteine and glutathione thereby confers resistance to proteasome inhibitor-mediated cytotoxicity (4). We showed that xCT expression is regulated by ARE and ATF4 binding motif amino acid response element (AARE) in the promoter region. In addition, ATF4 also binds to ARE in the 2nd intron in an Nrf2-depedent manner, most probably making loop formation between the intron and the promoter. We surmise that these interaction in the promoter is important for the cooperative activation of xCT gene transcription. Another cooperation between Nrf2 and ATF4 is observed using carnosic acid that is a major polyphenol contained in the herb rosemary (5). Carnosic acid only activates Nrf2 at its lower concentration, but at its higher concentration also activates ATF4 and invokes huge induction of Nrf2 target genes such as xCT, ?-glutamyl cysteine ligase and nerve growth factor and the induction is dependent both on Nrf2 and ATF4. We also interested in the interaction of Nrf2 pathway and the mitochondria, both are involved in the redox homeostasis during aging.
B. Elucidation of the Role of Nrf2 in the vasculature and the heart Nrf2 is activated by not only by oxidative stress but also by vascular shear stress and regulates vascular redox homeostasis by inducing expression of antioxidant proteins and enzymes (6). Nrf2 activity in the endothelium is regulated by laminar shear stress such that Nrf2 is high in the straight blood vessel, but low in the curvature or branching points of the blood vessels. Nrf2 and downstream antioxidant genes are thought to function as safeguards against various oxidative stress in general tissues, and of note, Nrf2 activity decreases with age. Nrf2 ameliorates endothelial dysfunction in diabetes and oxidative stress and increases the amount of bioavailable nitric oxide (NO), which is a major vasodilator. As the heart consists of postmitotic cells that utilize ATP produced mainly by mitochondrial oxidative phosphorylation (OXPHOS), cardiomyocytes appear to be equipped with highly sophisticated mitochondrial quality control mechanisms. Consistent with these findings, it has been reported that Nrf2 in the heart is regulated via a specific translational mechanism and that Nrf2 activation confers cardioprotective effects in various disease models. We will explore the role of Nrf2 in the heart.
C. Elucidation of the Role of Nrf2 in age-related diseases In contrast to the well-established activation mechanisms in response to extrinsic and endogenously generated electrophiles, it is not well understood whether Nrf2 responds to endogenous reactive oxygen species (ROS) generated in the mitochondria and whether Nrf2 is involved in the regulation of redox homeostasis of the organelle (5). As mitochondrial function and ROS generation are impaired with aging, it is important to understand the relationship of mitochondrial perturbation and Nrf2 activity, especially as they relate to the mitochondrial ROS. Furthermore, mitochondrial ROS and mitochondrial dysfunction play important roles in diverse age-related diseases and in physiology (7). We will explore the mechanisms of crosstalk between Nrf2 and mitochondria and their potential role in aging and age-related heart disease.
References
1. Discovery of the negative regulator of Nrf2, Keap1: a historical overview. Itoh K, Mimura J, Yamamoto M. Antioxid Redox Signal. 2010 Dec 1;13(11):1665-78.
2. Emerging Regulatory Role of Nrf2 in Iron, Heme, and Hemoglobin Metabolism in Physiology and Disease. Kasai S, Mimura J, Ozaki T, Itoh K. Front Vet Sci. 2018 Oct 10;5:242.
3. Nrf2- and ATF4-dependent upregulation of xCT modulates the sensitivity of T24 bladder carcinoma cells to proteasome inhibition. Ye P, Mimura J, Okada T, Sato H, Liu T, Maruyama A, Ohyama C, Itoh K. Mol Cell Biol. 2014 Sep 15;34(18):3421-34.
4. Concomitant Nrf2- and ATF4-activation by Carnosic Acid Cooperatively Induces Expression of Cytoprotective Genes. Mimura J, Inose-Maruyama A, Taniuchi S, Kosaka K, Yoshida H, Yamazaki H, Kasai S, Harada N, Kaufman RJ, Oyadomari S, Itoh K. Int J Mol Sci. 2019 Apr 5;20(7). pii: E1706.
5. Regulation of Nrf2 by Mitochondrial Reactive Oxygen Species in Physiology and Pathology. Kasai S, Shimizu S, Tatara Y, Mimura J, Itoh K. Biomolecules. 2020 Feb 17;10(2). pii: E320.
6. Emerging evidence for crosstalk between Nrf2 and mitochondria in physiological homeostasis and in heart disease. Tsushima M, Liu J, Hirao W, Yamazaki H, Tomita H, Itoh K. Arch Pharm Res. 2020 Mar;43(3):286-296.
7. Role of Mitochondrial Reactive Oxygen Species in the Activation of Cellular Signals, Molecules, and Function. Indo HP, Hawkins CL, Nakanishi I, Matsumoto KI, Matsui H, Suenaga S, Davies MJ, St Clair DK, Ozawa T, Majima HJ. Handb Exp Pharmacol. 2017;240:439-456.
Oxidative stress has been implicated in the pathogenesis of many age-related diseases such as Alzheimer's disease, age-related macular degeneration, and diabetes. Goal of this project is to develop a strategy for preventing age-related diseases targeting Nrf2-mediated cytoprotective mechanisms. Nrf2 is transcription factor that forms heterodimer with small Maf proteins to bind its target sequence antioxidant responsive element (ARE) (1, 2). Normally, Nrf2 is trapped to the E3 ligase adaptor Kelch-like ECH-associated protein 1 (Keap1) protein and subsequently degraded by the proteasome. However, when reactive cysteine residues of Keap1 protein are modified by oxidative stress or electrophilic substances, degradation of Nrf2 is inhibited, and accumulated Nrf2 translocates to the nucleus to bind to the ARE.
A. Elucidation of Nrf2 transcriptional network To elucidate the transcriptional network mediated by Nrf2, we previously performed yeast two hybrid screening using the C-terminus of Nrf2 that contains basic leucine zipper region of Nrf2. Interestingly, most positive clones encoded ATF4 (unpublished data, see also ref 3). Nrf2 and ATF4 are activated by the proteasome inhibition, cooperatively activates xCT expression which increases the intracellular cysteine and glutathione thereby confers resistance to proteasome inhibitor-mediated cytotoxicity (4). We showed that xCT expression is regulated by ARE and ATF4 binding motif amino acid response element (AARE) in the promoter region. In addition, ATF4 also binds to ARE in the 2nd intron in an Nrf2-depedent manner, most probably making loop formation between the intron and the promoter. We surmise that these interaction in the promoter is important for the cooperative activation of xCT gene transcription. Another cooperation between Nrf2 and ATF4 is observed using carnosic acid that is a major polyphenol contained in the herb rosemary (5). Carnosic acid only activates Nrf2 at its lower concentration, but at its higher concentration also activates ATF4 and invokes huge induction of Nrf2 target genes such as xCT, ?-glutamyl cysteine ligase and nerve growth factor and the induction is dependent both on Nrf2 and ATF4. We also interested in the interaction of Nrf2 pathway and the mitochondria, both are involved in the redox homeostasis during aging.
B. Elucidation of the Role of Nrf2 in the vasculature and the heart Nrf2 is activated by not only by oxidative stress but also by vascular shear stress and regulates vascular redox homeostasis by inducing expression of antioxidant proteins and enzymes (6). Nrf2 activity in the endothelium is regulated by laminar shear stress such that Nrf2 is high in the straight blood vessel, but low in the curvature or branching points of the blood vessels. Nrf2 and downstream antioxidant genes are thought to function as safeguards against various oxidative stress in general tissues, and of note, Nrf2 activity decreases with age. Nrf2 ameliorates endothelial dysfunction in diabetes and oxidative stress and increases the amount of bioavailable nitric oxide (NO), which is a major vasodilator. As the heart consists of postmitotic cells that utilize ATP produced mainly by mitochondrial oxidative phosphorylation (OXPHOS), cardiomyocytes appear to be equipped with highly sophisticated mitochondrial quality control mechanisms. Consistent with these findings, it has been reported that Nrf2 in the heart is regulated via a specific translational mechanism and that Nrf2 activation confers cardioprotective effects in various disease models. We will explore the role of Nrf2 in the heart.
C. Elucidation of the Role of Nrf2 in age-related diseases In contrast to the well-established activation mechanisms in response to extrinsic and endogenously generated electrophiles, it is not well understood whether Nrf2 responds to endogenous reactive oxygen species (ROS) generated in the mitochondria and whether Nrf2 is involved in the regulation of redox homeostasis of the organelle (5). As mitochondrial function and ROS generation are impaired with aging, it is important to understand the relationship of mitochondrial perturbation and Nrf2 activity, especially as they relate to the mitochondrial ROS. Furthermore, mitochondrial ROS and mitochondrial dysfunction play important roles in diverse age-related diseases and in physiology (7). We will explore the mechanisms of crosstalk between Nrf2 and mitochondria and their potential role in aging and age-related heart disease.
References
1. Discovery of the negative regulator of Nrf2, Keap1: a historical overview. Itoh K, Mimura J, Yamamoto M. Antioxid Redox Signal. 2010 Dec 1;13(11):1665-78.
2. Emerging Regulatory Role of Nrf2 in Iron, Heme, and Hemoglobin Metabolism in Physiology and Disease. Kasai S, Mimura J, Ozaki T, Itoh K. Front Vet Sci. 2018 Oct 10;5:242.
3. Nrf2- and ATF4-dependent upregulation of xCT modulates the sensitivity of T24 bladder carcinoma cells to proteasome inhibition. Ye P, Mimura J, Okada T, Sato H, Liu T, Maruyama A, Ohyama C, Itoh K. Mol Cell Biol. 2014 Sep 15;34(18):3421-34.
4. Concomitant Nrf2- and ATF4-activation by Carnosic Acid Cooperatively Induces Expression of Cytoprotective Genes. Mimura J, Inose-Maruyama A, Taniuchi S, Kosaka K, Yoshida H, Yamazaki H, Kasai S, Harada N, Kaufman RJ, Oyadomari S, Itoh K. Int J Mol Sci. 2019 Apr 5;20(7). pii: E1706.
5. Regulation of Nrf2 by Mitochondrial Reactive Oxygen Species in Physiology and Pathology. Kasai S, Shimizu S, Tatara Y, Mimura J, Itoh K. Biomolecules. 2020 Feb 17;10(2). pii: E320.
6. Emerging evidence for crosstalk between Nrf2 and mitochondria in physiological homeostasis and in heart disease. Tsushima M, Liu J, Hirao W, Yamazaki H, Tomita H, Itoh K. Arch Pharm Res. 2020 Mar;43(3):286-296.
7. Role of Mitochondrial Reactive Oxygen Species in the Activation of Cellular Signals, Molecules, and Function. Indo HP, Hawkins CL, Nakanishi I, Matsumoto KI, Matsui H, Suenaga S, Davies MJ, St Clair DK, Ozawa T, Majima HJ. Handb Exp Pharmacol. 2017;240:439-456.