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Multiple roles of Nrf2-Keap1 signaling a
Huai Deng a
Department of Biological Chemistry; University of Michigan Medical School; Ann Arbor, MI USA Published online: 01 Nov 2013.
Click for updates To cite this article: Huai Deng (2014) Multiple roles of Nrf2-Keap1 signaling, Fly, 8:1, 7-12, DOI: 10.4161/fly.27007 To link to this article: http://dx.doi.org/10.4161/fly.27007
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Fly 8:1, 7–12; January/February/March 2014; © 2014 Landes Bioscience
Multiple roles of Nrf2-Keap1 signaling Huai Deng
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Department of Biological Chemistry; University of Michigan Medical School; Ann Arbor, MI USA
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Keywords: xenobiotic response, development, transcriptional regulation, Nrf2/CncC, Keap1/dKeap1, steroid hormone, cancer Correspondence to: Huai Deng; E-mail: [email protected] Submitted: 09/25/2013 Revised: 10/28/2013 Accepted: 10/30/2013 http://dx.doi.org/10.4161/fly.27007 Extra view to: Deng H, Kerppola TK. Regulation of Drosophila metamorphosis by xenobiotic response regulators. PLoS Genet. 2013; 9; PMID: 23408904; http://dx.doi.org/10.1371/journal.pgen.1003263
enobiotic and oxidative responses protect cells from external and internal toxicities. Nrf2 and Keap1 are central factors that mediate these responses, and are closely related with many human diseases. In a recent study, we revealed novel developmental function and regulatory mechanism of Nrf2 and Keap1 by investigating their Drosophila homolog CncC and dKeap1. We found that CncC and dKeap1 control metamorphosis through regulations of ecdysone biosynthetic genes and ecdysone response genes in different tissues. CncC and dKeap1 cooperatively activate these developmental genes, in contrast to their conserved antagonizing effect to xenobiotic response transcription. In addition, interactions between CncC and Ras signaling in metamorphosis and in transcriptional regulation were established. Here I discuss the implications that place these classic xenobiotic response factors into a broader network that potentially control development and oncogenesis using mechanisms other than those mediating xenobiotic response.
as metabolism and development.3,4 Full elucidation of the molecular mechanisms and biological functions of these factors are important for understanding their roles in human health. The Nrf2-Keap1 signaling pathway plays a central role in xenobiotic and oxidative stress responses.5 Defects in this pathway are associated with diseases including cardiovascular defects, chronic inflammation, neurodegeneration, and cancer.2 Nrf2 (NF-E2-Related Factor 2) is a bZIP family transcription factor that can bind to and activate genes encoding detoxifying and antioxidant enzymes.6 Keap1 (Kelch-like ECH-Associated Protein 1) is a Kelch-family protein that inhibits the transcriptional activity of Nrf2. Keap1 can interact with Nrf2 and trigger its ubiquitination and proteasomal degradation in the cytoplasm. Interference of this interaction in response to stimuli leads to stabilization and nuclear accumulation of Nrf2.7,8 Mutations that eliminate the interaction between Nrf2 and Keap1 are associated with several cancers.9,10
Introduction
Drosophila Nrf2 and Keap1 Regulate Ecdysone Signaling
Conserved mechanisms mediate cell responses to a variety of xenobiotic compounds in the environment.1 These responses protect cells from the deleterious effects of xenobiotic compounds, while also rendering resistance to pharmacological treatments. Defects of these mechanisms are related to numerous pathological conditions.2 Some factors can mediate both responses to xenobiotic agents and normal cellular processes such
In a recently published study, we identified a novel developmental function of this classic xenobiotic response signaling.11 CncC and dKeap1, the homologs of Nrf2 and Keap1, mediate oxidative and xenobiotic responses in Drosophila.12,13 Immunostaining of polytene chromosomes revealed that both CncC and dKeap1 bind to ecdysone-regulated early puffs, indicating a relationship between the CncC-dKeap1 pathway and ecdysone
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Regulation of development and xenobiotic response using distinct mechanisms
signaling. Ecdysone is a steroid hormone that regulates multiple insect developmental programs.14 We then investigated the functions of CncC and dKeap1 in Drosophila development through tissuespecific knock down of these proteins. RNAi-depletion of CncC or dKeap1 in the salivary gland, an organ that displays ecdysone response, selectively reduces transcription of ecdysone-regulated early puff genes. Depletion of CncC or dKeap1 in the prothoracic gland, the organ that produces ecdysone, reduces ecdysone biosynthetic gene transcription, which consequently decreases ecdysteroid titer and delays pupation. These results establish the roles of CncC and dKeap1 in the regulation of transcriptional programs in different tissues that coordinate metamorphosis of Drosophila.
dKeap1 on Chromatin Studies mainly using cultured mammalian cells have shown that Keap1 predominantly localizes to the cytoplasm,
where Keap1 interacts with Nrf2 and induces its ubiquitination.7,8 A small portion of nuclear Keap1 has been revealed in mammalian cells,15 and was thought to transfer Nrf2 back to the cytoplasm after induction.16,17 However, ubiquitinated Nrf2 is prominently nuclear in HepG2 cells.16 We found that both dKeap1 and CncC predominantly localize to the nuclei of a number of Drosophila tissues,11 suggesting that dKeap1 interacts with CncC mainly in the nuclei. Therefore, Keap1/dKeap1 may regulate Nrf2/CncC using multiple mechanisms that are not mutually exclusive. Surprisingly, dKeap1 binds to specific loci on the polytene chromosomes. The direct binding and regulation of ecdysone response genes as well as ecdysone biosynthetic genes by dKeap1 suggest that this protein can function as a transcription factor. Both dKeap1 and CncC depletions reduced transcription of ecdysone biosynthetic and response genes, suggesting that dKeap1 and CncC can cooperatively regulate transcription. The co-localization of
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Regulation of Developmental Genes by Nrf2/CncC Our study indicated that CncC directly binds to and regulates ecdysone biosynthetic genes or ecdysone response genes in different tissues.11 A microarray study using adult flies that ectopically expressed CncC identified 127 genes that are associated with developmental processes.12 Poor overlap is found between the CncCregulatory profiles that are obtained by these 2 studies, except nvd and Sgs5. Neither of these 2 genes is activated by ectopic CncC in salivary glands (data not shown). The ecdysone biosynthetic genes are not expressed in salivary glands, and CncC does not occupy the loci encompassing these genes on polytene chromosomes (data not shown). Therefore, the
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Figure 1. Models for distinct transcriptional regulations by Nrf2/CncC and Keap1/dKeap1. The conserved Nrf2 and Keap1 family proteins can mediate transcriptional responses to both xenobiotic agents and developmental signals using distinct mechanisms. Left: Activation of xenobiotic response transcription by Nrf2/CncC is inhibited by the direct interaction with Keap1/dKeap1. Interference of this interaction by xenobiotic stimuli induces binding and activation of xenobiotic response genes by Nrf2/CncC. Middle: Activation of developmental genes by CncC is cooperated with chromatin-bound dKeap1. We hypothesize that the concerted activities of Nrf2/CncC and Keap1/dKeap1 can be regulated by xenobiotic compounds as well as developmental signals, which provides a mechanism that can adapt developmental programs to environmental chemicals. Right: Phosphorylation of CncC in response to Ras and possibly other signals can regulate chromatin binding specificity of CncC. This implicates another regulatory mechanism whereby Nrf2/CncC mediates the physiological functions and oncogenic effects of developmental signals.
dKeap1 and CncC at ecdysone-regulated puffs provides a hypothesis that the concerted chromatin binding of dKeap1 and CncC is required for transcriptional activation of target genes (Fig. 1). The targeting mechanisms and molecular functions of dKeap1 and CncC at these puffs are unknown. dKeap1 and CncC may participate in maintenance of open chromatin structure that facilitates transcription. It will be important to determine whether mammalian Keap1 also binds chromatin and regulates transcription in cooperation with Nrf2. A ChIP analysis in MDA-MB-231 cells did not detect Keap1 binding at the promoter of NQO-1, a xenobiotic response gene.17 Similarly, no dKeap1 occupancy was detected at loci encompassing conventional xenobiotic response genes on polytene chromosomes (data not shown). Opposite effects of dKeap1 and CncC on transcription of xenobiotic response genes were revealed in our study and also previously reported.11,12 This is consistent with the conserved function of Keap1/dKeap1 as suppressor of Nrf2/CncC. I hypothesize that Keap1 regulates xenobiotic response genes through inhibiting nuclear Nrf2 levels, while regulates some developmental genes through facilitating Nrf2 binding to chromatin (Fig. 1). Genome wide ChIP assays will determine the potential chromatin binding loci and target genes of Keap1 in mammalian cells.
cell proliferation.27 It is therefore likely that Nrf2 can distinctly regulate 2 types of genes: the xenobiotic response genes in inducible and global mode, and genes associated with normal cellular processes in basal and tissue specific mode. Tissue specific manipulation of Nrf2 or Keap1 family proteins in animal models will be necessary for elucidating their physiological functions. The mechanisms that coordinate the regulations of different categories of genes by Nrf2/CncC remain to be elucidated. It is likely that CncC/Nrf2 mediates transcriptional responses to developmental signals through mechanisms other than those that regulate xenobiotic responses. Given that dKeap1 binds to chromatin and regulates developmental genes in cooperation with CncC, Keap1/dKeap1 could directly control Nrf2/CncC activity at specific loci (Fig. 1). Another possible mechanism is the regulation of Nrf2/CncC binding specificity on chromatin, as supported by our observation that the CncC distribution on polytene chromosomes can be regulated by Ras signaling (Fig. 1).11 Relationships between Nrf2 and Ras/ ERK, insulin/PI3K/Akt, and mTOR pathways have been revealed recently.28,29 It will be interesting to examine whether the chromatin distribution Nrf2 and CncC can be regulated by these signaling pathways in both mammalian cells and Drosophila. Visualization of CncC and dKeap1 occupancies on polytene chromosomes provides an experimental system to test and search for internal or external signals that regulate the chromatin binding specificities of these factors.
Xenobiotic Response and Development Why are these xenobiotic response factors also employed to control developmental programs? One explanation is the evolutionarily relationship between genes that control xenobiotic response and genes that participate in the metabolism of endocrine hormones. Both Nrf2 and CncC can activate a number of cytochrome P450s,12,27 which belong to a superfamily of oxidoreductases that catalyze the metabolisms of many substances including lipids, vitamins, and steroid
hormones, as well as the detoxification of xenobiotic compounds.30 It is likely that the xenobiotic response genes and hormone metabolism genes evolved from the same families that were regulated by the same signaling pathway. Regulation of development by xenobiotic response factors can also serve as an adaptive function (Fig. 1). The development of many organisms is regulated by external factors, presumably to coordinate critical stages in the life cycle with favorable environmental conditions. Some mechanisms that regulate development in response to adverse conditions have been identified in Drosophila. Hypoxia reduces Drosophila body size by inhibiting somatic tissue growth, while it increases tracheal growth, both through HIF-1 signaling.31 Imaginal disc damage inhibits PTTH synthesis through a retinoid-controlled checkpoint, resulting in reduced ecdysone biosynthesis and delayed pupation.32 Damaged imaginal discs also secrete insulin-like peptide 8 (Dilp8), which delays metamorphosis through inhibiting both ecdysone biosynthesis and tissue growth.33,34 Modulation of TOR signaling in the prothoracic gland regulates ecdysone biosynthetic and ecdysone response gene transcription, as well as the timing of pupation. Activation of TOR signaling in the prothoracic gland suppresses the delay in pupation caused by larval starvation. These observations support a role of TOR signaling in the regulation of developmental timing in response to nutrient status.35 The effects of xenobiotic compounds have primarily been investigated using cultured cells and adult organisms. Nevertheless, many xenobiotic compounds can act as teratogens and disrupt the early development of mammals. Developing organisms are generally more sensitive to xenobiotic compounds than are mature or adult organisms. Drosophila is a valuable model to investigate the effects of xenobiotic compounds to different developmental programs, and to determine the roles of xenobiotic response factors in mediating these effects.
Nrf2, Ras, and Cancer The Nrf2-Keap1 pathway has essential, however contrasting effects on
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binding and regulation of developmental genes by CncC is tissue and stage specific. It is likely that the tissue specific regulation of genes that are unrelated to xenobiotic response is a common feature of the Nrf2 family proteins. For example, mouse Nrf2 can regulate lipid metabolism genes specifically in the liver.18 CncC overexpression markedly activates many xenobiotic response genes.12 However, few of the ecdysone response genes that are suppressed by CncC depletion are activated by CncC overexpression in salivary glands (data not shown). Consistently, ectopic CncC significantly binds to many loci on polytene chromosomes, but has a relatively low level of occupancy at early puffs. CncC can therefore maintain the basal expression of early puff genes and likely other developmental genes, but cannot activate these genes in response to xenobiotic stimuli. This provides an explanation for why the microarray analyses based on CncC overexpression failed to reveal most of the ecdysone response genes. Genome wide assays based on CncC depletion will comprehensively uncover developmental genes and functions that are regulated by this conserved xenobiotic response signaling. Recent studies have revealed Nrf2 target genes and functions other than detoxification and oxidant regulation. Targeted deletion of Nrf2 in mice causes developmental defects in adipogenesis and hematopoiesis, in addition to many oxidative stress defects upon treatment with xenobiotic agents.19–23 Nrf2 can directly bind and activate adipogenic genes such as peroxisome proliferator-activated receptor γ (Pparγ)21 and retinoid X receptor α (RXRA).24 Nrf2 controls hematopoietic stem cell functions through transcriptional regulation of CXCR4.22 In mouse liver, Nrf2 activates small heterodimer partner (SHP) nuclear receptor and lipid metabolism genes.18,25 In human lung cancer A549 cells, Nrf2 can activate glucose metabolism genes that facilitate cell proliferation, suggesting a novel mechanism of Nrf2 in promotion of carcinogenesis.26 ChIP-seq and microarray analyses using mouse embryonic fibroblasts with either enhanced or reduced levels of Nrf2 have identified around 1000 Nrf2-target genes, more than half of which were involved in
suggests that CncC mediates the function of Ras in ecdysone biosynthesis.11 This finding implicates a potential crosstalk among Ras, Nrf2, and steroid hormones. ChIP-seq and microarray analyses reveal that Nrf2 targets and regulates Cyp1b1 and Cyp39a1, both of which encode P450 enzymes that participate in metabolism of human steroid hormones.27,30 Nrf2 can mediate the 1α,25-dihydroxyvitamin D3-induced differentiation of acute myeloid leukemia cells through controlling VDR/RXRα transcription.44 Steroid hormones and their nuclear receptors are highly correlated with cancers and have been investigated as anticancer targets.45 It will be interesting to examine if mammalian Nrf2 regulates the productions of as well as the responses to steroid hormones, and if the interaction between Nrf2 and steroid hormone signaling contributes to carcinogenesis. Recent studies have revealed other downstream genes and functions of Nrf2 in facilitating cancer cell proliferation. Nrf2 can prevent apoptosis through activating the anti-apoptotic factor Bcl-2,46 control cell cycle through retinoblastoma protein,47 and facilitate cancer cell growth through influencing glucose metabolism.26 It is noticeable that Bcl-2, retinoblastoma, and glucose metabolism can all be regulated by Ras signaling,48 which further supports the interaction between Nrf2 and Ras in carcinogenesis. We found that CncC mediates the Ras-activation of Cp1 and Cyp28c1 genes that are unrelated to both xenobiotic response and ecdysone signaling.11 These studies predict additional genes and functions that are regulated by Ras-Nrf2/CncC pathway, indicating the necessity of genome wide analyses. Constitutive Ras signaling promotes carcinogenesis in Drosophila by facilitating both cell proliferation and cell migration.49,50 It will be interesting to test if CncC mediates these Ras-induced oncogenic events, taking advantage of the tissue specific genetic tools of Drosophila model.
Conclusion Since the discovery of Nrf2 and Keap1 factors more than a decade ago, most research focused on their functions in
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xenobiotic and oxidative responses. In recent years, increasing numbers of studies revealed the functions of these proteins in normal cellular processes in different phyla, providing novel insights to their roles in human health. Extensive investigations of the mechanisms that regulate Nrf2 and Keap1 family proteins as well as the complete range of their biological functions will be important for the development of therapeutic approaches against related diseases. Drosophila is a powerful model for many human diseases including cancer.51 The advantages of Drosophila genetics and imaging will provide contributions toward these goals. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
I thank Dr Tom Kerppola for critical comments and suggestions. I thank Anna Maurer for proofreading of the manuscript. This work was supported by the National Institute on Drug Abuse (T.K. DA030339) and by a fellowship from the University of Michigan Center for Organogenesis to H.D. References 1.
Jennings P, Limonciel A, Felice L, Leonard MO. An overview of transcriptional regulation in response to toxicological insult. Arch Toxicol 2013; 87:4972; PMID:22926699; http://dx.doi.org/10.1007/ s00204-012-0919-y 2. Sykiotis GP, Bohmann D. Stress-activated cap’n’collar transcription factors in aging and human disease. Sci Signal 2010; 3:re3; PMID:20215646; http://dx.doi. org/10.1126/scisignal.3112re3 3. Schmidt JV, Su GH, Reddy JK, Simon MC, Bradfield CA. Characterization of a murine Ahr null allele: involvement of the Ah receptor in hepatic growth and development. Proc Natl Acad Sci U S A 1996; 93:6731-6; PMID:8692887; http://dx.doi. org/10.1073/pnas.93.13.6731 4. Kastner P, Mark M, Ghyselinck N, Krezel W, Dupé V, Grondona JM, Chambon P. Genetic evidence that the retinoid signal is transduced by heterodimeric RXR/ RAR functional units during mouse development. Development 1997; 124:313-26; PMID:9053308 5. Slocum SL, Kensler TW. Nrf2: control of sensitivity to carcinogens. Arch Toxicol 2011; 85:27384; PMID:21369766; http://dx.doi.org/10.1007/ s00204-011-0675-4 6. Venugopal R, Jaiswal AK. Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductase1 gene. Proc Natl Acad Sci U S A 1996; 93:14960-5; PMID:8962164; http://dx.doi.org/10.1073/pnas.93.25.14960
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carcinogenesis. On one hand, Nrf2 deficient mice have increased susceptibility to chemical carcinogens since the xenobiotic response protects cells from their deleterious effects.5 Chemoprevention effects of Nrf2 inducers such as sulforaphane and CDDO-Im have been studied in both animal models and clinical trials.36,37 On the other hand, high levels of Nrf2 as well as mutations that are predicted to disrupt Nrf2-Keap1 complex are found in several human cancers.9,10 The mechanisms that contribute to the role of Nrf2 in promoting cancer are poorly understood. Genetic interactions between Nrf2/CncC and Ras oncogenic signaling were revealed in both mouse and Drosophila. Pancreatic and lung tumorigenesis induced by constitutively active K-RasG12D are suppressed in Nrf2 deficient mice.38 In Drosophila, Ras signaling regulates ecdysone biosynthesis in the prothoracic gland.39 We found that the premature pupation and developmental arrest caused by expression of constitutively active RasV12 in the prothoracic gland can be suppressed by CncC depletion.11 These studies suggest that the Nrf2/CncC can mediate the functions of Ras signaling in development and diseases including cancer. The molecular mechanisms whereby Ras regulates Nrf2/CncC remain to be established. Several evidences suggest that Ras/MAPK pathways can phosphorylate Nrf2 and control transcription activity or nuclear localization of Nrf2.40,41 In C. elegans, p38 MAPK catalyzes the phosphorylation of Nrf2 homolog SKN-1, which mediates its nuclear accumulation and transcription activity in response to oxidative stress.42 Nevertheless, phosphorylation of Nrf2 by MAPK only moderately enhance Nrf2 nuclear accumulation in 293T cells,43 suggesting that phosphorylation may control the Nrf2 activity through mechanisms other than regulation of its nuclear accumulation. Our discoveries that Ras controls the binding specificity of CncC at different loci, and that Ras and CncC regulate target genes in concert, suggest a model that phosphorylation can regulate the distribution and gene targeting of Nrf2/CncC factors on chromatin (Fig. 1). Genetic interaction between CncC and RasV12 in the regulation of metamorphosis
Itoh K, Wakabayashi N, Katoh Y, Ishii T, Igarashi K, Engel JD, Yamamoto M. Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes Dev 1999; 13:76-86; PMID:9887101; http:// dx.doi.org/10.1101/gad.13.1.76 8. Kobayashi A, Kang MI, Okawa H, Ohtsuji M, Zenke Y, Chiba T, Igarashi K, Yamamoto M. Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2. Mol Cell Biol 2004; 24:71309; PMID:15282312; http://dx.doi.org/10.1128/ MCB.24.16.7130-7139.2004 9. Padmanabhan B, Tong KI, Ohta T, Nakamura Y, Scharlock M, Ohtsuji M, Kang MI, Kobayashi A, Yokoyama S, Yamamoto M. Structural basis for defects of Keap1 activity provoked by its point mutations in lung cancer. Mol Cell 2006; 21:689700; PMID:16507366; http://dx.doi.org/10.1016/j. molcel.2006.01.013 10. Taguchi K, Motohashi H, Yamamoto M. Molecular mechanisms of the Keap1–Nrf2 pathway in stress response and cancer evolution. Genes Cells 2011; 16:123-40; PMID:21251164; http://dx.doi. org/10.1111/j.1365-2443.2010.01473.x 11. Deng H, Kerppola TK. Regulation of Drosophila metamorphosis by xenobiotic response regulators. PLoS Genet 2013; 9:e1003263; PMID:23408904; http://dx.doi.org/10.1371/journal.pgen.1003263 12. Misra JR, Horner MA, Lam G, Thummel CS. Transcriptional regulation of xenobiotic detoxification in Drosophila. Genes Dev 2011; 25:1796806; PMID:21896655; http://dx.doi.org/10.1101/ gad.17280911 13. Sykiotis GP, Bohmann D. Keap1/Nrf2 signaling regulates oxidative stress tolerance and lifespan in Drosophila. Dev Cell 2008; 14:76-85; PMID:18194654; http://dx.doi.org/10.1016/j. devcel.2007.12.002 14. Dubrovsky EB. Hormonal cross talk in insect development. Trends Endocrinol Metab 2005; 16:6-11; PMID:15620543; http://dx.doi.org/10.1016/j. tem.2004.11.003 15. Watai Y, Kobayashi A, Nagase H, Mizukami M, McEvoy J, Singer JD, Itoh K, Yamamoto M. Subcellular localization and cytoplasmic complex status of endogenous Keap1. Genes Cells 2007; 12:1163-78; PMID:17903176; http://dx.doi. org/10.1111/j.1365-2443.2007.01118.x 16. Nguyen T, Sherratt PJ, Nioi P, Yang CS, Pickett CB. Nrf2 controls constitutive and inducible expression of ARE-driven genes through a dynamic pathway involving nucleocytoplasmic shuttling by Keap1. J Biol Chem 2005; 280:32485-92; PMID:16000310; http://dx.doi.org/10.1074/jbc.M503074200 17. Sun Z, Zhang S, Chan JY, Zhang DD. Keap1 controls postinduction repression of the Nrf2-mediated antioxidant response by escorting nuclear export of Nrf2. Mol Cell Biol 2007; 27:6334-49; PMID:17636022; http://dx.doi.org/10.1128/MCB.00630-07 18. Kitteringham NR, Abdullah A, Walsh J, Randle L, Jenkins RE, Sison R, Goldring CE, Powell H, Sanderson C, Williams S, et al. Proteomic analysis of Nrf2 deficient transgenic mice reveals cellular defence and lipid metabolism as primary Nrf2-dependent pathways in the liver. J Proteomics 2010; 73:161231; PMID:20399915; http://dx.doi.org/10.1016/j. jprot.2010.03.018 19. Chan K, Lu R, Chang JC, Kan YW. NRF2, a member of the NFE2 family of transcription factors, is not essential for murine erythropoiesis, growth, and development. Proc Natl Acad Sci U S A 1996; 93:13943-8; PMID:8943040; http://dx.doi. org/10.1073/pnas.93.24.13943
20. Ma Q, Battelli L, Hubbs AF. Multiorgan autoimmune inflammation, enhanced lymphoproliferation, and impaired homeostasis of reactive oxygen species in mice lacking the antioxidant-activated transcription factor Nrf2. Am J Pathol 2006; 168:196074; PMID:16723711; http://dx.doi.org/10.2353/ ajpath.2006.051113 21. Pi J, Leung L, Xue P, Wang W, Hou Y, Liu D, Yehuda-Shnaidman E, Lee C, Lau J, Kurtz TW, et al. Deficiency in the nuclear factor E2-related factor-2 transcription factor results in impaired adipogenesis and protects against diet-induced obesity. J Biol Chem 2010; 285:9292-300; PMID:20089859; http://dx.doi.org/10.1074/jbc.M109.093955 22. Tsai JJ, Dudakov JA, Takahashi K, Shieh JH, Velardi E, Holland AM, Singer NV, West ML, Smith OM, Young LF, et al. Nrf2 regulates haematopoietic stem cell function. Nat Cell Biol 2013; 15:30916; PMID:23434824; http://dx.doi.org/10.1038/ ncb2699 23. Xue P, Hou Y, Chen Y, Yang B, Fu J, Zheng H, Yarborough K, Woods CG, Liu D, Yamamoto M, et al. Adipose deficiency of Nrf2 in ob/ob mice results in severe metabolic syndrome. Diabetes 2013; 62:84554; PMID:23238296; http://dx.doi.org/10.2337/ db12-0584 24. Chorley BN, Campbell MR, Wang X, Karaca M, Sambandan D, Bangura F, Xue P, Pi J, Kleeberger SR, Bell DA. Identification of novel NRF2-regulated genes by ChIP-Seq: influence on retinoid X receptor alpha. Nucleic Acids Res 2012; 40:7416-29; PMID:22581777; http://dx.doi.org/10.1093/nar/gks409 25. Huang J, Tabbi-Anneni I, Gunda V, Wang L. Transcription factor Nrf2 regulates SHP and lipogenic gene expression in hepatic lipid metabolism. Am J Physiol Gastrointest Liver Physiol 2010; 299:G1211-21; PMID:20930048; http://dx.doi. org/10.1152/ajpgi.00322.2010 26. Mitsuishi Y, Taguchi K, Kawatani Y, Shibata T, Nukiwa T, Aburatani H, Yamamoto M, Motohashi H. Nrf2 redirects glucose and glutamine into anabolic pathways in metabolic reprogramming. Cancer Cell 2012; 22:66-79; PMID:22789539; http:// dx.doi.org/10.1016/j.ccr.2012.05.016 27. Malhotra D, Portales-Casamar E, Singh A, Srivastava S, Arenillas D, Happel C, Shyr C, Wakabayashi N, Kensler TW, Wasserman WW, et al. Global mapping of binding sites for Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network analysis. Nucleic Acids Res 2010; 38:571834; PMID:20460467; http://dx.doi.org/10.1093/ nar/gkq212 28. Shibata T, Saito S, Kokubu A, Suzuki T, Yamamoto M, Hirohashi S. Global downstream pathway analysis reveals a dependence of oncogenic NF-E2related factor 2 mutation on the mTOR growth signaling pathway. Cancer Res 2010; 70:9095-105; PMID:21062981; http://dx.doi.org/10.1158/00085472.CAN-10-0384 29. Wang X, Tao L, Hai CX. Redox-regulating role of insulin: the essence of insulin effect. Mol Cell Endocrinol 2012; 349:111-27; PMID:21878367; http://dx.doi.org/10.1016/j.mce.2011.08.019 30. Guengerich FP. Cytochrome P450s and other enzymes in drug metabolism and toxicity. AAPS J 2006; 8:E101-11; PMID:16584116; http://dx.doi. org/10.1208/aapsj080112 31. Harrison JF, Haddad GG. Effects of oxygen on growth and size: synthesis of molecular, organismal, and evolutionary studies with Drosophila melanogaster. Annu Rev Physiol 2011; 73:95-113; PMID:20936942; http://dx.doi.org/10.1146/ annurev-physiol-012110-142155
32. Halme A, Cheng M, Hariharan IK. Retinoids regulate a developmental checkpoint for tissue regeneration in Drosophila. Curr Biol 2010; 20:458-63; PMID:20189388; http://dx.doi.org/10.1016/j. cub.2010.01.038 33. Garelli A, Gontijo AM, Miguela V, Caparros E, Dominguez M. Imaginal discs secrete insulin-like peptide 8 to mediate plasticity of growth and maturation. Science 2012; 336:579-82; PMID:22556250; http://dx.doi.org/10.1126/science.1216735 34. Colombani J, Andersen DS, Léopold P. Secreted peptide Dilp8 coordinates Drosophila tissue growth with developmental timing. Science 2012; 336:5825; PMID:22556251; http://dx.doi.org/10.1126/ science.1216689 35. Layalle S, Arquier N, Léopold P. The TOR pathway couples nutrition and developmental timing in Drosophila. Dev Cell 2008; 15:568-77; PMID:18854141; http://dx.doi.org/10.1016/j. devcel.2008.08.003 36. Cheung KL, Kong AN. Molecular targets of dietary phenethyl isothiocyanate and sulforaphane for cancer chemoprevention. AAPS J 2010; 12:8797; PMID:20013083; http://dx.doi.org/10.1208/ s12248-009-9162-8 37. Liby K, Royce DB, Williams CR, Risingsong R, Yore MM, Honda T, Gribble GW, Dmitrovsky E, Sporn TA, Sporn MB. The synthetic triterpenoids CDDO-methyl ester and CDDO-ethyl amide prevent lung cancer induced by vinyl carbamate in A/J mice. Cancer Res 2007; 67:2414-9; PMID:17363558; http://dx.doi.org/10.1158/00085472.CAN-06-4534 38. DeNicola GM, Karreth FA, Humpton TJ, Gopinathan A, Wei C, Frese K, Mangal D, Yu KH, Yeo CJ, Calhoun ES, et al. Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis. Nature 2011; 475:106-9; PMID:21734707; http://dx.doi.org/10.1038/nature10189 39. Rewitz KF, Yamanaka N, Gilbert LI, O’Connor MB. The insect neuropeptide PTTH activates receptor tyrosine kinase torso to initiate metamorphosis. Science 2009; 326:1403-5; PMID:19965758; http:// dx.doi.org/10.1126/science.1176450 40. Huang HC, Nguyen T, Pickett CB. Phosphorylation of Nrf2 at Ser-40 by protein kinase C regulates antioxidant response element-mediated transcription. J Biol Chem 2002; 277:42769-74; PMID:12198130; http://dx.doi.org/10.1074/jbc.M206911200 41. Zipper LM, Mulcahy RT. Erk activation is required for Nrf2 nuclear localization during pyrrolidine dithiocarbamate induction of glutamate cysteine ligase modulatory gene expression in HepG2 cells. Toxicol Sci 2003; 73:124-34; PMID:12657749; http://dx.doi.org/10.1093/toxsci/kfg083 42. Inoue H, Hisamoto N, An JH, Oliveira RP, Nishida E, Blackwell TK, Matsumoto K. The C. elegans p38 MAPK pathway regulates nuclear localization of the transcription factor SKN-1 in oxidative stress response. Genes Dev 2005; 19:2278-83; PMID:16166371; http://dx.doi.org/10.1101/ gad.1324805 43. Sun Z, Huang Z, Zhang DD. Phosphorylation of Nrf2 at multiple sites by MAP kinases has a limited contribution in modulating the Nrf2-dependent antioxidant response. PLoS One 2009; 4:e6588; PMID:19668370; http://dx.doi.org/10.1371/journal.pone.0006588 44. Bobilev I, Novik V, Levi I, Shpilberg O, Levy J, Sharoni Y, Studzinski GP, Danilenko M. The Nrf2 transcription factor is a positive regulator of myeloid differentiation of acute myeloid leukemia cells. Cancer Biol Ther 2011; 11:317-29; PMID:21099366; http://dx.doi.org/10.4161/cbt.11.3.14098
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47. Homma S, Ishii Y, Morishima Y, Yamadori T, Matsuno Y, Haraguchi N, Kikuchi N, Satoh H, Sakamoto T, Hizawa N, et al. Nrf2 enhances cell proliferation and resistance to anticancer drugs in human lung cancer. Clin Cancer Res 2009; 15:3423-32; PMID:19417020; http://dx.doi.org/10.1158/10780432.CCR-08-2822 48. Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D. RAS oncogenes: weaving a tumorigenic web. Nat Rev Cancer 2011; 11:761-74; PMID:21993244; http:// dx.doi.org/10.1038/nrc3106
49. Karim FD, Rubin GM. Ectopic expression of activated Ras1 induces hyperplastic growth and increased cell death in Drosophila imaginal tissues. Development 1998; 125:1-9; PMID:9389658 50. Pagliarini RA, Xu T. A genetic screen in Drosophila for metastatic behavior. Science 2003; 302:122731; PMID:14551319; http://dx.doi.org/10.1126/ science.1088474 51. Gilbert LI. Drosophila is an inclusive model for human diseases, growth and development. Mol Cell Endocrinol 2008; 293:25-31; PMID:18374475; http://dx.doi.org/10.1016/j.mce.2008.02.009
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45. Ahmad N, Kumar R. Steroid hormone receptors in cancer development: a target for cancer therapeutics. Cancer Lett 2011; 300:1-9; PMID:20926181; http:// dx.doi.org/10.1016/j.canlet.2010.09.008 46. Niture SK, Jaiswal AK. Nrf2 protein up-regulates antiapoptotic protein Bcl-2 and prevents cellular apoptosis. J Biol Chem 2012; 287:9873-86; PMID:22275372; http://dx.doi.org/10.1074/jbc. M111.312694
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Multiple roles of Nrf2-Keap1 signaling a
Huai Deng a
Department of Biological Chemistry; University of Michigan Medical School; Ann Arbor, MI USA Published online: 01 Nov 2013.
Click for updates To cite this article: Huai Deng (2014) Multiple roles of Nrf2-Keap1 signaling, Fly, 8:1, 7-12, DOI: 10.4161/fly.27007 To link to this article: http://dx.doi.org/10.4161/fly.27007
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Multiple roles of Nrf2-Keap1 signaling Huai Deng
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Department of Biological Chemistry; University of Michigan Medical School; Ann Arbor, MI USA
X
Keywords: xenobiotic response, development, transcriptional regulation, Nrf2/CncC, Keap1/dKeap1, steroid hormone, cancer Correspondence to: Huai Deng; E-mail: [email protected] Submitted: 09/25/2013 Revised: 10/28/2013 Accepted: 10/30/2013 http://dx.doi.org/10.4161/fly.27007 Extra view to: Deng H, Kerppola TK. Regulation of Drosophila metamorphosis by xenobiotic response regulators. PLoS Genet. 2013; 9; PMID: 23408904; http://dx.doi.org/10.1371/journal.pgen.1003263
enobiotic and oxidative responses protect cells from external and internal toxicities. Nrf2 and Keap1 are central factors that mediate these responses, and are closely related with many human diseases. In a recent study, we revealed novel developmental function and regulatory mechanism of Nrf2 and Keap1 by investigating their Drosophila homolog CncC and dKeap1. We found that CncC and dKeap1 control metamorphosis through regulations of ecdysone biosynthetic genes and ecdysone response genes in different tissues. CncC and dKeap1 cooperatively activate these developmental genes, in contrast to their conserved antagonizing effect to xenobiotic response transcription. In addition, interactions between CncC and Ras signaling in metamorphosis and in transcriptional regulation were established. Here I discuss the implications that place these classic xenobiotic response factors into a broader network that potentially control development and oncogenesis using mechanisms other than those mediating xenobiotic response.
as metabolism and development.3,4 Full elucidation of the molecular mechanisms and biological functions of these factors are important for understanding their roles in human health. The Nrf2-Keap1 signaling pathway plays a central role in xenobiotic and oxidative stress responses.5 Defects in this pathway are associated with diseases including cardiovascular defects, chronic inflammation, neurodegeneration, and cancer.2 Nrf2 (NF-E2-Related Factor 2) is a bZIP family transcription factor that can bind to and activate genes encoding detoxifying and antioxidant enzymes.6 Keap1 (Kelch-like ECH-Associated Protein 1) is a Kelch-family protein that inhibits the transcriptional activity of Nrf2. Keap1 can interact with Nrf2 and trigger its ubiquitination and proteasomal degradation in the cytoplasm. Interference of this interaction in response to stimuli leads to stabilization and nuclear accumulation of Nrf2.7,8 Mutations that eliminate the interaction between Nrf2 and Keap1 are associated with several cancers.9,10
Introduction
Drosophila Nrf2 and Keap1 Regulate Ecdysone Signaling
Conserved mechanisms mediate cell responses to a variety of xenobiotic compounds in the environment.1 These responses protect cells from the deleterious effects of xenobiotic compounds, while also rendering resistance to pharmacological treatments. Defects of these mechanisms are related to numerous pathological conditions.2 Some factors can mediate both responses to xenobiotic agents and normal cellular processes such
In a recently published study, we identified a novel developmental function of this classic xenobiotic response signaling.11 CncC and dKeap1, the homologs of Nrf2 and Keap1, mediate oxidative and xenobiotic responses in Drosophila.12,13 Immunostaining of polytene chromosomes revealed that both CncC and dKeap1 bind to ecdysone-regulated early puffs, indicating a relationship between the CncC-dKeap1 pathway and ecdysone
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Regulation of development and xenobiotic response using distinct mechanisms
signaling. Ecdysone is a steroid hormone that regulates multiple insect developmental programs.14 We then investigated the functions of CncC and dKeap1 in Drosophila development through tissuespecific knock down of these proteins. RNAi-depletion of CncC or dKeap1 in the salivary gland, an organ that displays ecdysone response, selectively reduces transcription of ecdysone-regulated early puff genes. Depletion of CncC or dKeap1 in the prothoracic gland, the organ that produces ecdysone, reduces ecdysone biosynthetic gene transcription, which consequently decreases ecdysteroid titer and delays pupation. These results establish the roles of CncC and dKeap1 in the regulation of transcriptional programs in different tissues that coordinate metamorphosis of Drosophila.
dKeap1 on Chromatin Studies mainly using cultured mammalian cells have shown that Keap1 predominantly localizes to the cytoplasm,
where Keap1 interacts with Nrf2 and induces its ubiquitination.7,8 A small portion of nuclear Keap1 has been revealed in mammalian cells,15 and was thought to transfer Nrf2 back to the cytoplasm after induction.16,17 However, ubiquitinated Nrf2 is prominently nuclear in HepG2 cells.16 We found that both dKeap1 and CncC predominantly localize to the nuclei of a number of Drosophila tissues,11 suggesting that dKeap1 interacts with CncC mainly in the nuclei. Therefore, Keap1/dKeap1 may regulate Nrf2/CncC using multiple mechanisms that are not mutually exclusive. Surprisingly, dKeap1 binds to specific loci on the polytene chromosomes. The direct binding and regulation of ecdysone response genes as well as ecdysone biosynthetic genes by dKeap1 suggest that this protein can function as a transcription factor. Both dKeap1 and CncC depletions reduced transcription of ecdysone biosynthetic and response genes, suggesting that dKeap1 and CncC can cooperatively regulate transcription. The co-localization of
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Regulation of Developmental Genes by Nrf2/CncC Our study indicated that CncC directly binds to and regulates ecdysone biosynthetic genes or ecdysone response genes in different tissues.11 A microarray study using adult flies that ectopically expressed CncC identified 127 genes that are associated with developmental processes.12 Poor overlap is found between the CncCregulatory profiles that are obtained by these 2 studies, except nvd and Sgs5. Neither of these 2 genes is activated by ectopic CncC in salivary glands (data not shown). The ecdysone biosynthetic genes are not expressed in salivary glands, and CncC does not occupy the loci encompassing these genes on polytene chromosomes (data not shown). Therefore, the
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Figure 1. Models for distinct transcriptional regulations by Nrf2/CncC and Keap1/dKeap1. The conserved Nrf2 and Keap1 family proteins can mediate transcriptional responses to both xenobiotic agents and developmental signals using distinct mechanisms. Left: Activation of xenobiotic response transcription by Nrf2/CncC is inhibited by the direct interaction with Keap1/dKeap1. Interference of this interaction by xenobiotic stimuli induces binding and activation of xenobiotic response genes by Nrf2/CncC. Middle: Activation of developmental genes by CncC is cooperated with chromatin-bound dKeap1. We hypothesize that the concerted activities of Nrf2/CncC and Keap1/dKeap1 can be regulated by xenobiotic compounds as well as developmental signals, which provides a mechanism that can adapt developmental programs to environmental chemicals. Right: Phosphorylation of CncC in response to Ras and possibly other signals can regulate chromatin binding specificity of CncC. This implicates another regulatory mechanism whereby Nrf2/CncC mediates the physiological functions and oncogenic effects of developmental signals.
dKeap1 and CncC at ecdysone-regulated puffs provides a hypothesis that the concerted chromatin binding of dKeap1 and CncC is required for transcriptional activation of target genes (Fig. 1). The targeting mechanisms and molecular functions of dKeap1 and CncC at these puffs are unknown. dKeap1 and CncC may participate in maintenance of open chromatin structure that facilitates transcription. It will be important to determine whether mammalian Keap1 also binds chromatin and regulates transcription in cooperation with Nrf2. A ChIP analysis in MDA-MB-231 cells did not detect Keap1 binding at the promoter of NQO-1, a xenobiotic response gene.17 Similarly, no dKeap1 occupancy was detected at loci encompassing conventional xenobiotic response genes on polytene chromosomes (data not shown). Opposite effects of dKeap1 and CncC on transcription of xenobiotic response genes were revealed in our study and also previously reported.11,12 This is consistent with the conserved function of Keap1/dKeap1 as suppressor of Nrf2/CncC. I hypothesize that Keap1 regulates xenobiotic response genes through inhibiting nuclear Nrf2 levels, while regulates some developmental genes through facilitating Nrf2 binding to chromatin (Fig. 1). Genome wide ChIP assays will determine the potential chromatin binding loci and target genes of Keap1 in mammalian cells.
cell proliferation.27 It is therefore likely that Nrf2 can distinctly regulate 2 types of genes: the xenobiotic response genes in inducible and global mode, and genes associated with normal cellular processes in basal and tissue specific mode. Tissue specific manipulation of Nrf2 or Keap1 family proteins in animal models will be necessary for elucidating their physiological functions. The mechanisms that coordinate the regulations of different categories of genes by Nrf2/CncC remain to be elucidated. It is likely that CncC/Nrf2 mediates transcriptional responses to developmental signals through mechanisms other than those that regulate xenobiotic responses. Given that dKeap1 binds to chromatin and regulates developmental genes in cooperation with CncC, Keap1/dKeap1 could directly control Nrf2/CncC activity at specific loci (Fig. 1). Another possible mechanism is the regulation of Nrf2/CncC binding specificity on chromatin, as supported by our observation that the CncC distribution on polytene chromosomes can be regulated by Ras signaling (Fig. 1).11 Relationships between Nrf2 and Ras/ ERK, insulin/PI3K/Akt, and mTOR pathways have been revealed recently.28,29 It will be interesting to examine whether the chromatin distribution Nrf2 and CncC can be regulated by these signaling pathways in both mammalian cells and Drosophila. Visualization of CncC and dKeap1 occupancies on polytene chromosomes provides an experimental system to test and search for internal or external signals that regulate the chromatin binding specificities of these factors.
Xenobiotic Response and Development Why are these xenobiotic response factors also employed to control developmental programs? One explanation is the evolutionarily relationship between genes that control xenobiotic response and genes that participate in the metabolism of endocrine hormones. Both Nrf2 and CncC can activate a number of cytochrome P450s,12,27 which belong to a superfamily of oxidoreductases that catalyze the metabolisms of many substances including lipids, vitamins, and steroid
hormones, as well as the detoxification of xenobiotic compounds.30 It is likely that the xenobiotic response genes and hormone metabolism genes evolved from the same families that were regulated by the same signaling pathway. Regulation of development by xenobiotic response factors can also serve as an adaptive function (Fig. 1). The development of many organisms is regulated by external factors, presumably to coordinate critical stages in the life cycle with favorable environmental conditions. Some mechanisms that regulate development in response to adverse conditions have been identified in Drosophila. Hypoxia reduces Drosophila body size by inhibiting somatic tissue growth, while it increases tracheal growth, both through HIF-1 signaling.31 Imaginal disc damage inhibits PTTH synthesis through a retinoid-controlled checkpoint, resulting in reduced ecdysone biosynthesis and delayed pupation.32 Damaged imaginal discs also secrete insulin-like peptide 8 (Dilp8), which delays metamorphosis through inhibiting both ecdysone biosynthesis and tissue growth.33,34 Modulation of TOR signaling in the prothoracic gland regulates ecdysone biosynthetic and ecdysone response gene transcription, as well as the timing of pupation. Activation of TOR signaling in the prothoracic gland suppresses the delay in pupation caused by larval starvation. These observations support a role of TOR signaling in the regulation of developmental timing in response to nutrient status.35 The effects of xenobiotic compounds have primarily been investigated using cultured cells and adult organisms. Nevertheless, many xenobiotic compounds can act as teratogens and disrupt the early development of mammals. Developing organisms are generally more sensitive to xenobiotic compounds than are mature or adult organisms. Drosophila is a valuable model to investigate the effects of xenobiotic compounds to different developmental programs, and to determine the roles of xenobiotic response factors in mediating these effects.
Nrf2, Ras, and Cancer The Nrf2-Keap1 pathway has essential, however contrasting effects on
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binding and regulation of developmental genes by CncC is tissue and stage specific. It is likely that the tissue specific regulation of genes that are unrelated to xenobiotic response is a common feature of the Nrf2 family proteins. For example, mouse Nrf2 can regulate lipid metabolism genes specifically in the liver.18 CncC overexpression markedly activates many xenobiotic response genes.12 However, few of the ecdysone response genes that are suppressed by CncC depletion are activated by CncC overexpression in salivary glands (data not shown). Consistently, ectopic CncC significantly binds to many loci on polytene chromosomes, but has a relatively low level of occupancy at early puffs. CncC can therefore maintain the basal expression of early puff genes and likely other developmental genes, but cannot activate these genes in response to xenobiotic stimuli. This provides an explanation for why the microarray analyses based on CncC overexpression failed to reveal most of the ecdysone response genes. Genome wide assays based on CncC depletion will comprehensively uncover developmental genes and functions that are regulated by this conserved xenobiotic response signaling. Recent studies have revealed Nrf2 target genes and functions other than detoxification and oxidant regulation. Targeted deletion of Nrf2 in mice causes developmental defects in adipogenesis and hematopoiesis, in addition to many oxidative stress defects upon treatment with xenobiotic agents.19–23 Nrf2 can directly bind and activate adipogenic genes such as peroxisome proliferator-activated receptor γ (Pparγ)21 and retinoid X receptor α (RXRA).24 Nrf2 controls hematopoietic stem cell functions through transcriptional regulation of CXCR4.22 In mouse liver, Nrf2 activates small heterodimer partner (SHP) nuclear receptor and lipid metabolism genes.18,25 In human lung cancer A549 cells, Nrf2 can activate glucose metabolism genes that facilitate cell proliferation, suggesting a novel mechanism of Nrf2 in promotion of carcinogenesis.26 ChIP-seq and microarray analyses using mouse embryonic fibroblasts with either enhanced or reduced levels of Nrf2 have identified around 1000 Nrf2-target genes, more than half of which were involved in
suggests that CncC mediates the function of Ras in ecdysone biosynthesis.11 This finding implicates a potential crosstalk among Ras, Nrf2, and steroid hormones. ChIP-seq and microarray analyses reveal that Nrf2 targets and regulates Cyp1b1 and Cyp39a1, both of which encode P450 enzymes that participate in metabolism of human steroid hormones.27,30 Nrf2 can mediate the 1α,25-dihydroxyvitamin D3-induced differentiation of acute myeloid leukemia cells through controlling VDR/RXRα transcription.44 Steroid hormones and their nuclear receptors are highly correlated with cancers and have been investigated as anticancer targets.45 It will be interesting to examine if mammalian Nrf2 regulates the productions of as well as the responses to steroid hormones, and if the interaction between Nrf2 and steroid hormone signaling contributes to carcinogenesis. Recent studies have revealed other downstream genes and functions of Nrf2 in facilitating cancer cell proliferation. Nrf2 can prevent apoptosis through activating the anti-apoptotic factor Bcl-2,46 control cell cycle through retinoblastoma protein,47 and facilitate cancer cell growth through influencing glucose metabolism.26 It is noticeable that Bcl-2, retinoblastoma, and glucose metabolism can all be regulated by Ras signaling,48 which further supports the interaction between Nrf2 and Ras in carcinogenesis. We found that CncC mediates the Ras-activation of Cp1 and Cyp28c1 genes that are unrelated to both xenobiotic response and ecdysone signaling.11 These studies predict additional genes and functions that are regulated by Ras-Nrf2/CncC pathway, indicating the necessity of genome wide analyses. Constitutive Ras signaling promotes carcinogenesis in Drosophila by facilitating both cell proliferation and cell migration.49,50 It will be interesting to test if CncC mediates these Ras-induced oncogenic events, taking advantage of the tissue specific genetic tools of Drosophila model.
Conclusion Since the discovery of Nrf2 and Keap1 factors more than a decade ago, most research focused on their functions in
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xenobiotic and oxidative responses. In recent years, increasing numbers of studies revealed the functions of these proteins in normal cellular processes in different phyla, providing novel insights to their roles in human health. Extensive investigations of the mechanisms that regulate Nrf2 and Keap1 family proteins as well as the complete range of their biological functions will be important for the development of therapeutic approaches against related diseases. Drosophila is a powerful model for many human diseases including cancer.51 The advantages of Drosophila genetics and imaging will provide contributions toward these goals. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
I thank Dr Tom Kerppola for critical comments and suggestions. I thank Anna Maurer for proofreading of the manuscript. This work was supported by the National Institute on Drug Abuse (T.K. DA030339) and by a fellowship from the University of Michigan Center for Organogenesis to H.D. References 1.
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carcinogenesis. On one hand, Nrf2 deficient mice have increased susceptibility to chemical carcinogens since the xenobiotic response protects cells from their deleterious effects.5 Chemoprevention effects of Nrf2 inducers such as sulforaphane and CDDO-Im have been studied in both animal models and clinical trials.36,37 On the other hand, high levels of Nrf2 as well as mutations that are predicted to disrupt Nrf2-Keap1 complex are found in several human cancers.9,10 The mechanisms that contribute to the role of Nrf2 in promoting cancer are poorly understood. Genetic interactions between Nrf2/CncC and Ras oncogenic signaling were revealed in both mouse and Drosophila. Pancreatic and lung tumorigenesis induced by constitutively active K-RasG12D are suppressed in Nrf2 deficient mice.38 In Drosophila, Ras signaling regulates ecdysone biosynthesis in the prothoracic gland.39 We found that the premature pupation and developmental arrest caused by expression of constitutively active RasV12 in the prothoracic gland can be suppressed by CncC depletion.11 These studies suggest that the Nrf2/CncC can mediate the functions of Ras signaling in development and diseases including cancer. The molecular mechanisms whereby Ras regulates Nrf2/CncC remain to be established. Several evidences suggest that Ras/MAPK pathways can phosphorylate Nrf2 and control transcription activity or nuclear localization of Nrf2.40,41 In C. elegans, p38 MAPK catalyzes the phosphorylation of Nrf2 homolog SKN-1, which mediates its nuclear accumulation and transcription activity in response to oxidative stress.42 Nevertheless, phosphorylation of Nrf2 by MAPK only moderately enhance Nrf2 nuclear accumulation in 293T cells,43 suggesting that phosphorylation may control the Nrf2 activity through mechanisms other than regulation of its nuclear accumulation. Our discoveries that Ras controls the binding specificity of CncC at different loci, and that Ras and CncC regulate target genes in concert, suggest a model that phosphorylation can regulate the distribution and gene targeting of Nrf2/CncC factors on chromatin (Fig. 1). Genetic interaction between CncC and RasV12 in the regulation of metamorphosis
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