Multiple roles of Nrf2-Keap1 signaling

Abstract

Xenobiotic 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.

Introduction

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 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

Drosophila Nrf2 and Keap1 Regulate Ecdysone Signaling

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 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 tissue-specific 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 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.

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.

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.

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 CncC-regulatory 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 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 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 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-Ras^G12D 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 Ras^V12 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 Ras^V12 in the regulation of metamorphosis 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 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.

Deng H, Kerppola TK. Regulation of Drosophila metamorphosis by xenobiotic response regulators. . PLoS Genet. 2013;9:e1003263. doi: 10.1371/journal.pgen.1003263.

Submitted

09/25/2013

Revised

10/28/2013

Accepted

10/30/2013

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.