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Regulation of Notch1 Signaling by Nrf2: Implications for Tissue Regeneration

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Science Signaling  13 Jul 2010:
Vol. 3, Issue 130, pp. ra52
DOI: 10.1126/scisignal.2000762


The Keap1-Nrf2-ARE signaling pathway elicits an adaptive response for cell survival after endogenous and exogenous stresses, such as inflammation and carcinogens, respectively. Keap1 inhibits the transcriptional activation activity of Nrf2 (p45 nuclear factor erythroid-derived 2–related factor 2) in unstressed cells by facilitating its degradation. Through transcriptional analyses in Keap1- or Nrf2-disrupted mice, we identified interactions between the Keap1-Nrf2-ARE and the Notch1 signaling pathways. We found that Nrf2 recognized a functional antioxidant response element (ARE) in the promoter of Notch1. Notch1 regulates processes such as proliferation and cell fate decisions. We report a functional role for this cross talk between the two pathways and show that disruption of Nrf2 impeded liver regeneration after partial hepatectomy and was rescued by reestablishment of Notch1 signaling.


Nrf2 (p45 nuclear factor erythroid-derived 2–related factor 2) is a prosurvival transcription factor that plays a pivotal role in maintaining cellular homeostasis after electrophile and oxidative stresses (1). Coordinated changes in response to Nrf2 activation result in enhanced expression of genes that contribute to (i) free radical metabolism, (ii) electrophile detoxication, (iii) glutathione biosynthesis, (iv) generation of reducing equivalents, (v) formation of direct antioxidants, (vi) toxin efflux transporters, (vii) the proteasomal system, and (viii) modulation of inflammatory pathways. Many of these genes contain cis elements in their regulatory domains known as antioxidant response elements (AREs) (2). The ARE interacts with the cap ‘n’ collar (Cnc) family of basic leucine zipper (bZip) transcription factors, including Nrf2, and their dimerization partners that include other bZip transcription factors, such as the small Mafs (1). Experiments with knockout mice for various genes encoding Cnc family members revealed that these transcription factors contribute to both constitutive and inducible expression of these cytoprotective genes (3).

Analysis of Nrf2-null mice revealed that Nrf2 is the primary factor in regulation of gene expression through the ARE in vivo. The knockout mice survive and produce viable offspring, but they are more sensitive toward toxicities associated with various electrophiles and reactive oxygen species than are wild-type mice (3, 4). In early development, Nrf2-null mice appear normal. However, it has not been determined whether development in the knockout mice is altered under stressed conditions. By contrast, mice null for Nrf1 (encoding a related Cnc bZip transcription factor) show a drastic phenotype in early development and are embryonic-lethal at mid-to-late gestation as a result of anemia caused by a non–cell-autonomous defect in erythropoiesis in the fetal liver (5). This observation suggests a fundamental role for Nrf1 in development. Analysis of the liver-specific Nrf1 conditional knockout mouse indicates that Nrf1 contributes to postnatal ARE-dependent gene regulation, as well as having a prenatal role (6). Because the Nrf1::Nrf2 double-knockout mouse has a shorter life span in utero than the Nrf1 single knockout, Nrf2 has been proposed to contribute or partially compensate as a transcription factor in early development (7). Nrf2 influences tissue injury and repair. The increased abundance of Nrf2 in hepatocyte-specific Keap1-disrupted mice results in resistance to chemical- and inflammation-mediated hepatotoxicity (8, 9). By contrast, Nrf2-null mice, which are more sensitive to hepatotoxins (10), exhibit significantly delayed liver regeneration after partial hepatectomy (11).

Differential microarray analyses have been conducted in several tissues of wild-type and Nrf2-null mice (12, 13) to help define cytoprotective pathways and to characterize the role of the Keap1-Nrf2-ARE pathway in the pharmacodynamic action of several classes of anticarcinogens (14, 15). However, these analyses reflect expression patterns from heterogeneous cell populations within the analyzed tissues. To overcome this limitation, we established immortalized mouse embryonic fibroblasts (MEFs) from Keap1−/−, Nrf2−/−, Keap1−/−::Nrf2−/−, and wild-type mice (16). These MEFs showed pronounced differential sensitivity to redox cycling quinones (17) and photooxidative damage from combined exposure to retinaldehyde and ultraviolet A light (18). Strong differential expression of Notch1 and some of its downstream effectors was observed, raising the possibility that the Notch1 signaling cascade may be regulated at the transcriptional level through interactions of Nrf2 with ARE. Levels of Notch1 messenger RNA (mRNA) were substantially reduced in Nrf2-null MEFs compared to wild type; transcripts for Notch1 target genes, such as Hes1, Herp1, Herp2 (19), and Nrarp (20), were also reduced significantly.

The Notch family of transmembrane receptors participates in a signaling pathway controlling a broad spectrum of metazoan cell fates and developmental processes through local cell-cell interactions (21). Alteration of signaling through the Notch family of receptors can markedly affect differentiation, proliferation, and apoptotic events. Genetic ablation studies indicate that Notch1 is crucial for early development and regrowth of several tissues (22, 23). Activation of the Notch pathway inhibits differentiation in different developmental contexts and has been associated with the amplification of some somatic stem cells—not only the neural (24) and hematopoietic stem cells (25) but also the hepatocyte (26, 27) and intestinal epithelial stem cells (28, 29). Considering the importance of the Notch1 signal cascade in developmental biology, the microarray observations indicated the possibility that Nrf2 could be a key molecule affecting both embryonic and adult tissue stem cell renewal, as well as cell fates. This study characterizes the effects of Nrf2 genotype on the expression of Notch1 and its effector genes, and the importance of Nrf2-Notch1 cross talk in liver regeneration.


Expression of Notch1 and its effector genes is decreased in Nrf2-null MEFs

Microarray analyses compared gene expression patterns in wild-type or Nrf2-disrupted mice to identify Nrf2-regulated genes (13) and provided hints for interactions between Nrf2 and other signaling pathways such as the aryl hydrocarbon receptor (AhR) (30) and nuclear factor κB (Nf-κB) pathways (31). Further analysis of one of these data sets (30) revealed that Notch1 transcript levels were 14-fold lower in Nrf2-null compared to wild-type MEFs. Furthermore, the expression of Hes1 and p21, two downstream genes in the Notch1 pathway (19, 32), was also reduced substantially in Nrf2-null cells. To confirm the microarray data indicating that Nrf2 promotes the Notch1 signaling pathway, we analyzed the expression of additional downstream effector genes by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) in wild-type and Nrf2-null MEFs. We found that the abundances of transcripts for the Notch1 target genes Hes1, Herp1, Herp2, Nrarp, and p21 were also reduced in Nrf2-null MEFs, corresponding to the reduced Notch1 expression. By contrast, no effect on the abundance of transcript for the Notch1 ligand Jag1 was observed (Fig. 1A). Quantitative real-time RT-PCR analysis confirmed the reduction in Notch1 expression in Nrf2-null mice (Fig. 1B). We also found that expression of a prototypic Nrf2-regulated gene, Gsta1, was substantially lower in the Nrf2-deficient fibroblasts. Expression of Gsta1 was induced by treatment with 2.5 μM sulforaphane (an activator of Nrf2 signaling) (14) in wild-type cells only (Fig. 1B). As with Gsta1, induction of Notch1 by sulforaphane was also lost in the absence of functional Nrf2 (Fig. 1B). These results suggest a role for Nrf2 in Notch1 expression.

Fig. 1

Differential expression of Notch1 and related genes in wild-type (Nrf2+/+) and Nrf2-null (Nrf2−/−) MEFs. (A) Semiquantitative RT-PCR for Notch1, Notch1 effector genes (Hes1, Herp1, Herp2, Nrarp, and p21), and Notch1 ligand-encoding gene (Jag1). The abundance of the transcript for Tata box–binding protein (TBP) served as a loading control. Data are quantified as the ratio of the expression of the indicated genes in Nrf2 null/wild type. Values are the means ± SE of three independent experiments. Expression of all effector genes, except Jag1, was significantly lower in the Nrf2-null MEFs (P < 0.01, t test). (B) Real-time RT-PCR confirmation of changes in Notch1 expression in wild-type versus Nrf2-null MEFs. The loss of induction of Gsta1, an Nrf2-responsive gene, confirmed the disruption in Nrf2 activity. MEF cultures were treated with 2.5 μM sulforaphane or vehicle 24 hours before cells were harvested for RNA isolation. Values are means ± SE (n = 3). *P < 0.05, t test. (C) RT-PCR analyses of Notch1 and other Nrf2 target gene expression in the livers from 8-week-old Nrf2-null, wild-type, and various Keap1 and Nrf2 compound-disrupted mice. Hypoxanthine-guanine phosphoribosyltransferase (Hprt) served as the loading control. For quantitation, see fig. S1, B and C.

Notch1 expression is influenced by Nrf2 abundance in mice

To elucidate whether the effect of Nrf2 genotype on Notch1 expression observed in MEFs also occurred in vivo, we assessed transcript abundance in adult livers. Notch1 transcripts were consistently reduced by ~40% in livers of adult Nrf2-null mice compared to those in wild-type mouse livers (Fig. 1C and fig. S1B). Keap1-null mice, which exhibit constitutive activation of the Nrf2 pathway, die by 3 weeks of age from malnutrition through formation of esophageal stricture (33). This knockout mouse can be rescued by reducing the function of Nrf2 through genetic engineering. To analyze the effect of Nrf2 on Notch1 expression in adult mice, we measured Notch1 transcripts in livers of 8-week-old male mice of three genotypes: Keap1−/−::Nrf2+/−, Keap1+/−::Nrf2+/−, and Keap1+/−::Nrf2−/−. Although the mice of these three genotypes survive, the amount of functional Nrf2 differs among the genotypes. In the Keap1-deficient mice, which partially or completely lack this repressor of Nrf2, Nrf2 activity is higher in Keap1−/−::Nrf2+/− than in Keap1+/−::Nrf2+/− mice (33, 34). Nrf2 is absent in Keap1+/−::Nrf2−/− mice. The order of abundance of hepatic Nrf2 is Keap1−/−::Nrf2+/− > Keap1+/−::Nrf2+/− > Keap1+/−::Nrf2−/−. Notch1 expression exhibits the same qualitative pattern by genotype, as does expression of two other Nrf2 target genes, Nqo1 and Gsta1 (Fig. 1C and fig. S1C). Thus, the magnitude of Nrf2 signaling appears to affect the extent of Notch1 expression.

The regulatory region of the Notch1 gene contains functional ARE

We investigated the possibility that Notch1 is a direct target gene of the Nrf2 transcription factor. Sequence analysis [National Center for Biotechnology Information mouse genome sequence viewer and (35)] revealed that the proximal promoter region of Notch1 bears four consensus AREs (shown as 1 to 4 in Fig. 2A). Forced expression of Nrf2 enhanced the activity of the −1640 Notch1 luciferase reporter gene (Fig. 2B) in MEF cells. Also, in accord with the observations in MEF cells, treatment with sulforaphane significantly increased Notch1 reporter activity in the presence and absence of exogenous Nrf2. Thus, Nrf2 appears to directly affect Notch1 expression in MEFs, and this is likely mediated by binding of Nrf2 to regulatory domains in the −1640 proximal promoter of Notch1.

Fig. 2

Analysis of the proximal promoter of murine Notch1 in MEFs. A ~2-kb portion of the promoter of Notch1 was isolated from murine liver and ligated into a luciferase reporter vector (−1640 Notch1-Luc) to monitor its activity. (A) Distribution of putative ARE motifs. (B) Effect of the presence of Nrf2 and sulforaphane (2.5 μM) treatment on −1640 Notch1-Luc reporter gene activity in MEFs. Values are means ± SE (n = 3). *P < 0.05. Data are shown as the relative luciferase activity (RLA) compared to that in vector (pCMVMock)–transfected cells in the absence of sulforaphane, which was set at a value of 1.0. (C) Serial deletion assays of the −1640 Notch1-Luc reporter with relative luciferase activities in MEF (left) and P19 EC (right) cells. In both analyses, the activities from the −78 construct were set at a value of 1.0. Black ellipses show the presence of putative AREs. Values are means ± SD (n = 3). Luciferase activities were normalized by measuring the Renilla luciferase activity from a cotransfected reporter vector. *P < 0.05. (D) Effect of site-directed mutagenesis on the enhancer capacity of the 1-ARE motif in MEFs. Cells were transfected either with the −206 Notch1-Luc truncated promoter construct (p-206 Luc), which contained 1-ARE, or with a form with point mutations in 1-ARE (p-206 mARE Luc). RLA was based on the activity from each −206 Luc reporter construct when transfected with the vector pCMVMock, which was set at a value of 1.0. Values are means ± SD. *P < 0.05. (E) Recombinant Nrf2 and MafK, produced as MBP fusion proteins, form a complex with Notch1–1-ARE, which can be competed away by Gsta1-ARE but not by mARE. Left panels show negative controls for the recombinant MBP-Nrf2 and MBP-MafK fusion proteins used for the EMSA analysis. Right panels show EMSA in the presence of the ARE. (F) Binding of nuclear factors from MEFs to the 1-ARE of Notch1 can be competed away with the ARE of the mouse Gsta1 promoter region (table S1), but not by Notch1 mARE. A 25-fold molar excess of each competitor was used. (G) The binding complex can be supershifted with an antibody to Nrf2, but not with an antibody to Nrf1 or with normal rabbit IgG. The arrowhead indicates the specific protein complex with the 1-ARE probe, and the arrow indicates the supershifted band. (H) ChIP assay using wild-type and Nrf2−/− cells. No DNA and IgG reactions were the controls for PCR and immunoprecipitation steps, respectively. The 1-ARE in the Notch1 gene-regulatory region was detected only in the presence of wild-type cell nuclear extract and the antibody to Nrf2. The Gsta1 promoter region, which has a functional ARE, served as a positive control.

To elucidate whether and where any functional enhancer regions of Notch1 are located, we transfected serial deletion reporter constructs bearing the Notch1 5′-flanking region into both wild-type MEF and the P19 embryonal carcinoma (EC) cell line, in which Notch1 is abundantly expressed during differentiation (36). We identified the region from −315 to −189 as the positive regulatory region in both cell types (Fig. 2C). One consensus ARE core sequence (1-ARE), TGABNNNGC (B is C, G, or T; N is A, T, G, or C) (1), was present in the region −204 to −196. To elucidate whether 1-ARE was functional, we created p-206 and point mutant reporter genes (Fig. 2D) and transfected them into MEFs. Coexpression of Nrf2 with p-206 led to a fourfold enhancement in reporter activity compared to that in vector-transfected cells (Fig. 2D). The responsiveness of a p-206 mutant ARE (mARE) luciferase reporter to Nrf2 was reduced relative to that of the wild-type p-206 reporter. These results suggest that 1-ARE in the Notch1 gene-regulatory region is a functional cis element in MEFs.

We performed a series of electrophoretic mobility shift assays (EMSAs) to determine whether the positive regulatory element directly binds transcriptional factors. There are three major Cnc candidates for binding to AREs and stimulating gene expression: Nrf1, Nrf2, and Nrf3 (37). Nrf3 was considered the least likely molecule because of its low expression among the three candidates in MEFs. Neither Nrf1 nor Nrf3 gene expression was altered in the wild-type compared to Nrf2−/− MEFs (fig. S1A). We considered Nrf2 the strongest candidate on the basis of the comparison of Notch1 expression in Nrf2-null versus wild-type MEFs. To determine whether Nrf2 formed a complex with 1-ARE from the Notch1 gene-regulatory region, we prepared recombinant Nrf2 and its binding partner, small MafK. Neither Nrf2 alone nor MafK alone bound the Notch1 1-ARE (Fig. 2E, left). Formation of the heterodimeric complex (Nrf2-MafK) bound to the Notch1 1-ARE occurred only when both proteins were added (Fig. 2E, right). The intensities of the bands representing complex formation with both recombinant proteins were reduced by the addition of a cold competitor oligonucleotide, the ARE from Gsta1, but not by a point mutant oligonucleotide of the Notch1 1-ARE. Thus, Notch1 1-ARE has cis element potential for Nrf2-MafK heterodimer binding.

1-ARE formed complexes when incubated with nuclear extracts from MEFs (Fig. 2F). The band indicating the binding complex disappeared after addition of the Gsta1-ARE as a competitor, but not in the presence of excess mutant Notch1 1-ARE (Fig. 2F). An EMSA conducted with an antibody to Nrf2 showed supershifted bands from the complex; however, we did not observe supershift or disappearance of the bands in the presence of an antibody to Nrf1 (Fig. 2G). To determine whether basal Notch1 expression is stimulated by Nrf2 through 1-ARE in MEFs, we performed a chromatin immunoprecipitation (ChIP) assay with wild-type and Nrf2−/− MEFs. Under stringent conditions in which the positive control Gsta1-ARE locus (38) was barely detectable, the antibody to Nrf2 precipitated the proximal region of the Notch1 promoter, including 1-ARE, in nuclear extracts from wild-type, but not Nrf2−/−, MEFs. Collectively, the luciferase reporter, EMSA, and ChIP results indicate that Nrf2, likely in complex with other bZip transcription factors such as the small Mafs, forms a complex with 1-ARE of the Notch1 gene-regulatory region.

Notch1 expression is increased in RosaCreER/CreER::Keap1flox/flox MEFs

In the Keap1−/− liver, Nrf2 accumulates in the nucleus as a result of impaired Keap1-mediated Nrf2 degradation. We found that the amount of Notch1 transcripts was increased in Keap1−/− P1 liver (fig. S1D). However, the constitutively active status of Nrf2 may produce indirect effects on gene expression. Indeed, these Keap1−/− mice do not survive more than 3 weeks (33). To avoid such constitutive Nrf2 activation and toxicity, we isolated conditional Nrf2-activating MEFs (RCKF) from RosaCreER/CreER::Keap1flox/flox mice, as well as control MEFs (RC) from RosaCreER/CreER mice. Keap1 was disrupted in RCKF cells exposed to 4-hydroxytamoxifen (4-OHT), but not when exposed to vehicle (Fig. 3A). In control RC MEFs, 4-OHT did not induce Notch1 expression or that of Nqo1, a representative Nrf2 target gene (Fig. 3B). Thus, 4-OHT does not itself influence Nrf2-related gene expression. Expression of both Nqo1 and Notch1 was induced in RCKFs treated with 4-OHT (Fig. 3B), and this was accompanied by the accumulation of Nrf2 (Fig. 3C) and disruption of Keap1 (Fig. 3A). Thus, alteration of the abundance of cellular Nrf2 directly influenced the abundance of Notch1 transcripts.

Fig. 3

The timing-specific induction of Notch1 by Nrf2 is shown with RosaCreER/CreER::Keap1flox/flox (RCKF) MEF cells. (A) Scheme of the genetic alleles present within the mouse constructs used: the mutant cell line carrying the floxed Keap1 gene (top) and the expected disrupted allele (bottom). RosaCreER/CreER MEF cells (RC1 and RC2), as well as RCKF1 and RCKF2 cells, were treated with 1 μM 4-OHT for 3 days with repetitive treatments every 24 hours. WT, wild type; D, disrupted gene; Un, undisrupted gene. (B) Treatment with 4-OHT induces Notch1 and Nqo1 gene expression in RCKF MEF cells, as shown by RT-PCR. The magnitude of the expression of each gene was normalized by the expression of the housekeeping gene Hprt using the total RNA derived from both RC1 and RCKF1 MEFs (right). Gene expression from vehicle-treated samples in each cell was set at a value of 1.0. Values are means ± SD (n = 3). *P < 0.01, one-way ANOVA. (C) Immunoblotting of Nrf2 in the RCKF MEF cell extracts shows that Nrf2 accumulates in cells after treatment with 4-OHT.

Notch1 signaling is reduced in Nrf2-null MEFs

We developed a coculture model to determine whether downstream Notch1 signaling was affected by Nrf2 genotype. Nrf2-disrupted or wild-type MEFs were each cocultured with clones of human embryonic kidney 293 (HEK293) cells stably overexpressing Notch1 ligands [human Delta-like 1 (DLL1) and human JAG1], which served as pathway activators (Fig. 4). We assessed the amount of the exogenous Notch1 ligands JAG1 and DLL1 in the HEK293 cells by immunoblot analysis (Fig. 4A). To evaluate the activity of the Notch1 pathway, we monitored the expression of the direct Notch1 target genes Hes1, Herp1, Herp2, and Nrarp. When wild-type MEFs were cocultured with J1 or J2 HEK293 cells, both of which stably express human JAG1 protein, we observed a significant increase in the expression of the Notch1 target genes compared to that in cocultures with clone 3 of mock-transfected HEK293 cells. Similar relative induction of Hes1, Herp1, Herp2, or Nrarp was seen when the J1 or J2 lines were cocultured with Nrf2−/− MEFs, although absolute expression was considerably lower than in wild-type MEFs. With the DLL1-expressing, D2-stable clone, Hes1 transcripts were increased in cocultures with wild-type MEFs but showed a dampened response in cocultures with Nrf2-null MEFs (Fig. 4C). Herp1, which is another direct target gene of Notch1, also exhibited increased transcript abundance in wild-type but not Nrf2-null MEFs cocultured with DLL1-overxpressing HEK293 cells. Thus, Nrf2-null MEFs show significantly decreased Notch responses.

Fig. 4

In MEFs, Notch1 signaling is dependent on Nrf2 genotype. (A) The abundance of Notch1 signaling ligands (DLL1 or JAG1) in independent lines of stably transfected HEK293 cells as shown by immunoblot analysis with JAG1- or DLL1-specific antibodies (sc-8303 and sc-9102). Probing the blots with a lamin B1 antibody (sc-6216) served as the loading control. M1 to M4, mock; D1 to D4, DLL1; J1 to J4, JAG1. (B) Effect of Nrf2 genotype on Notch1 signaling in MEFs cocultured with JAG1-expressing HEK293 (J2) cells. (C) Time course showing Notch1 signaling in MEF cells cocultured with DLL1-expressing HEK293 (D2) cells. Gene expression in (B) and (C) was examined by real-time RT-PCR. 18S ribosomal RNA expression was used for normalization. The expression for each gene in Nrf2+/+ MEFs was set at a value of 1.0. Values are means ± SD (n = 3). *P > 0.05, one-way ANOVA.

Nrf2−/− mice exhibit delayed liver regeneration and diminished Notch1 signaling

Notch signaling is essential during embryogenesis of mice. Conditional gene targeting studies have revealed that Notch signaling is also important after birth, as well as in adult mice (39, 40). Notch1 is activated and plays an important role in cell proliferation during liver regeneration after partial hepatectomy. Liver injection of silencing RNA for Notch1 or Jag1 before partial hepatectomy significantly suppresses the proliferation of hepatocytes at days 2 to 4 of the regenerative response (26). Liver regeneration from partial hepatectomy is also delayed in Nrf2-disrupted mice compared to that in wild-type mice (41). We confirmed this delayed liver regeneration at 72 hours after partial hepatectomy in Nrf2-null mice (Fig. 5C). We hypothesized that this delay was associated with altered Notch1 expression and signaling in the Nrf2-disrupted mice. To verify this hypothesis, we performed two-thirds partial hepatectomy (both median and left lobes) in Nrf2-null mice and measured the transcription of the Notch1 target gene Hes1 to evaluate the impact on Notch1 signaling (Fig. 5A). In livers of mice of each genotype, at 30 min after partial hepatectomy, the abundance of transcripts for Nqo1, which reflects Nrf2 activity, and Hes1, which reflects Notch1 signaling, was similar to the abundance before hepatectomy (fig. S2). However, at 3 hours after partial hepatectomy, there were increases in transcripts encoding the Notch1 ligands (Jag1 and Dll1) in livers of both genotypes (Fig. 5A). The abundance of the Notch1 transcript in livers of Nrf2−/− mice was less than the abundance in livers from wild-type mice, and the Nrf2−/− mice failed to induce Notch1 6 hours after partial hepatectomy (Fig. 5B). Hes1 transcripts were more abundant in the livers of wild-type mice compared to the amount in livers of Nrf2−/− mice. Although the relative induction of Hes1 was similar between genotypes, the absolute amount of Hes1 transcripts was lower after partial hepatectomy in Nrf2−/− mice (Fig. 5B). The amount of basal Nqo1 transcripts was also reduced in the Nrf2-null mice, and the induction in response to partial hepatectomy was completely blocked. In the livers of sham-operated mice, the abundance of transcripts for Rbpj, the direct partner of Notch1 for the activation of the target genes, was not different in the livers of Nrf2−/− mice compared to that in the wild-type mouse livers, and the abundance of these transcripts decreased 6 hours after partial hepatectomy only in the Nrf2−/− mice (Fig. 5B). This result suggests that the different responses of Nqo1 (absence of induction) and Hes1 (reduced induction) to partial hepatectomy between wild-type and Nrf2-null mice are caused by differences in initial amounts of Nrf2 and Notch1 proteins in the liver. The absence of Nrf2 in the liver seemed to affect Notch1 signaling in the early phase of the regenerative process after two-thirds partial hepatectomy because of the lower basal transcript abundance of Notch1 in the Nrf2-null mice.

Fig. 5

Reduced basal Notch1 signaling in Nrf2-null mice delays liver regeneration. (A) The expression of Jag1 and Dll1, genes encoding Notch1 ligands, in whole liver was analyzed by real-time RT-PCR 3 hours after two-thirds partial hepatectomy. Hprt expression was used for normalization. (B) Hes1 expression was monitored by real-time RT-PCR as an index of Notch1 signaling 6 hours after two-thirds partial hepatectomy. Albumin expression was used for normalization. The ratio of hepatic gene of interest to albumin gene expression in sham-operated Nrf2+/+ mice was set at a value of 1.0. Values are means ± SD (n = 3). *P < 0.05, one-way ANOVA. (C) Relative liver weights from Nrf2−/− and wild-type mice (age- and total body weight–matched) 3 days after two-thirds partial hepatectomy (PH). Values are means ± SD. *P < 0.01, one-way ANOVA. White boxes in (A) to (C) represent sham-operated animals; black boxes represent partial hepatectomy animals.

Hepatocyte-specific expression of the Notch intracellular domain rescued the delayed regeneration of the liver after partial hepatectomy in Nrf2-null mice

To restore Notch1 signaling in the hepatocytes in the livers of Nrf2−/− mice, we established triple-compound Nrf2−/−::RosaNICD/−::AlbCre mice by crossing Nrf2-null mice with RosaNICD/− or AlbCre transgenic mice in a step-by-step manner. Only in this composite line of mice are the Notch intracellular domain (NICD), which is the transcriptionally active Notch1 cleavage fragment, and enhanced green fluorescent protein (EGFP) in hepatocytes present because of the removal of the transcriptional stop signal sequence placed between both loxP sequences directly downstream of the Rosa 26 promoter region by the specific expression of Cre recombinase controlled by the albumin enhancer promoter (Fig. 6A). Homologous recombination in the Rosa 26 locus (Cre-active) leading to expression of NICD was detected only in the DNA isolated from the liver of Nrf2−/−::RosaNICD/−::AlbCre mice (Fig. 6B). Both NICD and EGFP were present only in the triple-compound mice, as detected by immunoblot analyses of whole-liver extracts (Fig. 6C). The abundance of transcripts for the Notch1 direct target gene Hes1 was increased in the Nrf2−/−::RosaNICD/−::AlbCre mice relative to the abundance in the other two lines of mice (Fig. 6D). Thus, this triple-compound line exhibited enhanced Notch1 signaling in hepatocytes. Two-thirds partial hepatectomies were conducted on this line and two sets of control lines, Nrf2−/−::RosaNICD/− and Nrf2−/−::AlbCre. There were no significant differences among three genotypes in the size of livers in animals undergoing sham partial hepatectomy (Fig. 6E). However, after partial hepatectomy, the Nrf2−/−::RosaNICD/−::AlbCre line showed enhanced regeneration of liver weight compared to that seen in wild-type mice (Fig. 6E, compare with Fig. 5B). Neither of the control lines (Nrf2−/−::RosaNICD/− or Nrf2−/−::AlbCre) influenced the delayed regeneration rate typical of the Nrf2-null mice. These results demonstrate that constitutive expression of the gene encoding Notch1 and Notch1 signaling are supported by Nrf2 and contribute to tissue regeneration.

Fig. 6

NICD restores liver regeneration in Nrf2-null mice. (A) Scheme for hepatocyte-specific NICD expression. Arrows show primers used for genotyping. Sequences are described in table S2. wt, wild-type locus; Tg, transgenic Rosa 26 locus. (B) Demonstration of homologous recombination only in genomic DNA isolated from the livers of Nrf2−/−::RosaNICD/−::AlbCre mice. mut, Nrf2-disrupted allele; Cre, AlbCre recombinase insertion allele. (C) Immunoblots of NICD and EGFP demonstrating that the proteins were present only in the liver of Nrf2−/−::RosaNICD/−::AlbCre mice. Lamin B1 served as the control for protein loading and was detected with an antibody. (D) Hepatic expression of Hes1 in Nrf2−/−::RosaNICD/−, Nrf2−/−::AlbCre, and Nrf2−/−::RosaNICD/−::AlbCre mice. The ratios (mean ± SD) of hepatic Hes1 expression are 0.58 ± 0.13 and 4.31 ± 0.72 in Nrf2−/−::RosaNICD/− and Nrf2−/−::RosaNICD/−::AlbCre, respectively. Hes1 expression in Nrf2−/−::AlbCre was set at a value of 1.0 (n = 3). P < 0.01, one-way ANOVA. (E) Regenerative recovery of liver mass in Nrf2−/−::RosaNICD/−, Nrf2−/−::AlbCre, and Nrf2−/−::RosaNICD/−::AlbCre mice. Values are means ± SD. *P < 0.01, one-way ANOVA. White boxes, sham-operated animals; black boxes, partial hepatectomy animals.


We provide several lines of evidence indicating that Nrf2 enhances the expression of the gene encoding Notch1 and the amplitude of its downstream signals (Fig. 1). First, a comparison of expression profiles in MEFs isolated from wild-type and Nrf2-disrupted mice showed that Notch1 expression was dampened in the Nrf2-null cells. Additionally, transcripts for downstream effectors of Notch1—Hes1, Herps, Nrarp, and p21—were also diminished in the knockout cells. Second, the Notch1 target genes were induced by treatment of wild-type, but not Nrf2-knockout, cells with the chemopreventive agent sulforaphane, an activator of Nrf2 signaling. Third, genetic manipulation of the abundance of Nrf2 in mouse liver—by disruption of the genes encoding Nrf2 and its repressor, Keap1—demonstrated a dose-dependent association between magnitude of Nrf2 signaling and Notch1 expression. Notch1 expression was decreased in the livers of Nrf2-null mice but was enhanced in Keap1−/−::Nrf2+/− mouse livers, which is similar to the expression of prototypic Nrf2-regulated genes Gsta1 and Nqo1 in these genotypic settings. Although the Keap1−/−::Nrf2+/− mice exhibit a constitutive activation of Nrf2, these animals do not exhibit the preweaning lethal phenotype of Keap1-null mice.

The coculture experiments with HEK293 cells overexpressing the Notch1 ligands JAG1 or DLL1 indicated that decreased expression of Notch1 in Nrf2-null MEF directly impaired Notch1 signal transduction, as reflected in reduced expression of its target genes Hes1, Herps, and Nrarp. Thus, Notch1 signaling is limited by the abundance of Notch1, which is transcriptionally controlled by Nrf2, not by the abundance of the Notch1 ligands. This ligand-Notch1 interaction may mimic events early in tissue regeneration after injury. Partial hepatectomy provides a model to examine signaling pathways involved in the response to hepatic injury. Previous studies have shown that Notch1 signaling increases rapidly after partial hepatectomy (26) and that liver regeneration is delayed in Nrf2-null mice (41), an observation that we confirm here. Köhler et al. (26) observed that nuclear translocation of NICD increased and peaked within 15 min after partial hepatectomy in rats, indicating activation of Notch signaling. In the same study, expression of the Notch-dependent target gene Hes1 increased within 30 to 60 min. We report that at 6 hours after partial hepatectomy, no change in abundance of Notch1 and Nrf2 transcripts was observed, but Hes1 transcripts, as well as those encoding the Notch1 ligands Jag1 or Dll1, were increased. Although we observed similar relative increases of Hes1 transcripts in the livers of Nrf2-null mice after partial hepatectomy, the absolute amount was only one-third of that in wild-type mice. This phenomenon was similar to the observations in the in vitro coculture system with MEFs. Because the abundance of the transcripts of the Notch1 ligands did not appear to be influenced by Nrf2 genotype after partial hepatectomy, the differential up-regulation of Hes1 by genotype may reflect abundance of Notch1 protein. Notch1 signaling, which is supported by Nrf2, appeared to influence early steps in liver regeneration, and we tested this by creating Nrf2−/−::RosaNICD/−::AlbCre mice, in which NICD is produced specifically in hepatocytes in the Nrf-null background. Forced hepatic Notch1 signaling mediated by NICD rescues the phenotype of delayed liver regeneration observed in the Nrf2-null mice.

Other effects of Nrf2 on Notch1 signaling can be envisioned. Nrf2-null mice do not show any growth and development phenotypes under normal conditions, either in early development or postnatally (3, 42). By contrast, Notch1 disruption is lethal to mice at around embryonic day 10.5 (E10.5), indicating that this gene and its signals are essential in embryonic development stage before E10.5 (21). The Nrf2 transcript is detected at this time only in the central nervous system by RNA in situ hybridization, and this is the earliest point of detection (43). Thus, Notch1 is likely present before Nrf2 (gene expression data from Mouse Genome Informatics), suggesting that expression of Notch1 in early development does not depend on Nrf2. Alternatively, considering Nrf1 expression patterns during embryonic development and the embryonic lethal phenotype of Nrf1-disrupted mice, Nrf1 may substitute for Nrf2 to support Notch1 expression through 1-ARE early in life. Further analyses are required to elucidate how ARE-containing genes are regulated by Nrf2, Nrf1, or other factors during embryogenesis.

Nrf2-mediated regulation of Notch1 expression may be most critical in postnatal stages, rather than during development, when Notch1 is likely regulated by other factors, because Nrf2 disruption does not produce altered phenotypes during early development stages. Nrf2 is abundant in liver, kidney, lung, and the gastrointestinal tract, tissues that also contain stem cells. Notch and Notch-related gene expression also occur in these tissues (26, 28, 29, 44, 45). In such tissues, damaged cells must be replaced by newly differentiated cells for routine tissue maintenance. Furthermore, many of these tissues face the highest burdens from environmental exposures to toxicants and resultant tissue injury. The Keap1-Nrf2 pathway regulates a well-described adaptive response that reduces macromolecular damage after exposure to conditions that increase the amounts of free radicals and electrophiles through stimulation of genes encoding antioxidative and electrophile detoxication enzymes (4). Nrf2 signaling also stimulates pathways affecting the recognition, repair, or removal, or all of these, of damaged macromolecules, notably proteins and DNA (38). Our previous work indicates that repair of tissue damage represents a third component of the adaptive cytoprotective response controlled by Nrf2. Nrf2-null mice are considerably more sensitive to hyperoxic lung injury (46). Treatment of mice with the triterpenoid Nrf2 activator 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO–imidazolide) during, but not before, hyperoxic exposure led to substantial protection against the biochemical and morphological sequelae of acute lung injury and appeared to enhance tissue remodeling (47). The current study indicates that Nrf2 facilitates tissue regeneration in the liver by regulating Notch1 expression. With these diverse actions influencing the cellular response to toxins or tissue damage, strategies using drugs or foods to enhance Nrf2 signaling and that of its interacting networks provide multifaceted opportunities for disease prevention.

Materials and Methods

Mice and genotyping

Keap1+/− mice were backcrossed >10 times with C57BL/6J mice purchased from the Jackson Laboratory. Nrf2−/− mice, which were backcrossed >20 times into a C57BL/6J background, were a gift from K. Itoh (Hirosaki University). Keap1-Nrf2 compound knockout mice were established by mating Keap1+/− with Nrf2+/− mice to generate Keap1+/−::Nrf2+/− mice. Each compound genotype was obtained from matings between Keap1+/−::Nrf2+/− mice (33). Keap1flox/+ mice were generated as described previously (8). Gt(ROSA)26Sortm1(Cre/Esr1)Nat mice (RosaCreER/CreER) (48), Gt(ROSA)26Sortm1(Notch1)Dam/J (RosaNICD/−) (49), and B6.Cg-Tg(Alb-cre)21Mgn/J (AlbCre) (50) were obtained from the Jackson Laboratory. Keap1flox/flox::RosaCreER/CreER mice were established by matings with Keap1flox/+ and RosaCreER/CreER mice in consecutive steps. Nrf2−/−::RosaNICD/−::AlbCre mice resulted from crossings with Nrf2−/−::RosaNICD/NICD and Nrf2−/−::AlbCre mice, which were established by the consecutive mating of Nrf2+/−::AlbCre and Nrf2+/−::RosaNICD/− mice. All mice were genotyped by previously reported methods (8, 33, 48) or by PCR described in table S2. All animal breeding and handling were conducted in accordance with protocols approved by the Animal Care and Use Committee of the Johns Hopkins Medical Institutions.

Partial hepatectomy

Male mice (9 to 10 weeks old) were placed into a Plexiglas chamber for induction of anesthesia with 2% isoflurane and oxygen flow (2 liters/min). After primary anesthetization, they were maintained under anesthesia by isoflurane inhalation through a suitable mouthpiece. The left and median lobes of the liver were pushed out from an incision just under the xiphoid process and removed with a ligature (51). After surgery, mice were returned to their cages and given free access to food and water. Sham-operated control animals were treated in an identical fashion with the omission of hepatectomy.

Cell culture

MEFs were established from the embryos of Nrf2-null or wild-type littermates. On day 14.5 of pregnancy, mice were killed and embryos were removed from the uterus. Extraembryonic tissues were removed from individual fetuses, followed by rinsing in phosphate-buffered saline (PBS) to remove any blood. Embryos were decapitated and eviscerated with sterile forceps. A portion of the tail was used as a source of DNA for genotype and gender confirmation (52) by PCR. The carcasses were transferred to a sterile conical centrifuge tube containing PBS and rinsed by inverting twice. The carcasses were dissociated by finely mincing with a scalpel blade and then pressing through a sterile cell strainer with forceps. They were rinsed on the screen twice with a 0.05% trypsin solution, transferred to a small culture flask, and stirred gently in a humidified 5% CO2 incubator at 37°C for 30 min. Suspended cells were decanted and collected by centrifugation. Cell pellets were washed twice with 30 ml of Iscove’s minimum essential medium (MEM, Invitrogen) containing 10% fetal bovine serum (FBS, Invitrogen) (MEF growth medium). Cells were plated on dishes in MEF growth medium; 24 hours later, the cells were rinsed with PBS–0.02% EDTA twice and the medium was replaced with fresh MEF growth medium. In about 2 to 4 days, the cultures were confluent and expanded for use in experiments and for making frozen stocks. MEFs derived from male embryos were used in all experiments. P19 cells (American Type Culture Collection) were grown as monolayers and maintained in α-MEM (Sigma) containing 10% FBS, and then incubated at 37°C in a humidified atmosphere of 5% CO2. To establish human JAG1– or human DLL1–expressing cells, we transfected pLZRShJagged1 or pLZRhDll1 (53), provided by L. Parreira (University of Lisbon), into HEK293 cells. Simultaneously, negative control transfectant, which was transfected with LZRSpBMN-linker-IRES-EGFP as a mock vector, was also prepared. After 2 weeks of selection with puromycin (10 μg/ml), several single resistant colonies were picked and maintained in each dish with Iscove’s MEM containing 10% FBS and puromycin (8 μg/ml). RCKF and RC MEFs were isolated from embryos of RosaCreER/CreER::Keap1flox/flox or RosaCreER/CreER pregnant mice (ED14.5), respectively. Each MEF culture was established from single embryos. Experiments were conducted within passage number 5. 4-OHT (Sigma) was dissolved in 100% ethanol at 10 mM. RCKF and RC were analyzed after 3 days of repetitive exposure to conditioned media that included 1 μM 4-OHT.

Coculture assay for Notch signaling

Cells expressing the Notch1 ligand or negative control cells (5 × 105) were spread on 10-cm dishes 4 days before coculture with Nrf2-null or wild-type MEFs. Nrf2-null or wild-type MEFs were plated at 4 × 105 cells per dish. Notch ligand–expressing cells or control cells were collected with PBS-EDTA (final, 0.02% EDTA) and washed once before seeding. The transfectants were split into dishes of each MEF genotype containing complete medium and incubated at 37°C in 5% CO2. Total RNA was isolated and analyzed by RT-PCR. It was confirmed that the primer set amplified only products from MEFs under our PCR conditions.

Protein preparation and immunoblot analyses

Proper size cut tissues were homogenized in radioimmunoprecipitation assay buffer (RIPA-I), which contained a protease inhibitor cocktail (Roche). Cultured cells were harvested with RIPA-I buffer directly. An equal volume of 2× SDS sample buffer was added, and the samples were denatured by boiling for 5 min. Samples were applied to SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to an Immobilon polyvinylidene difluoride membrane (Millipore). The membranes were blocked with tris-buffered saline containing 0.05% Tween 20 and 5% skim milk and then treated with a primary antibody. The preparative membranes were reacted with appropriate secondary antibodies conjugated to horseradish peroxidase (Invitrogen, Zymed). The immunocomplexes were visualized with enhanced chemiluminescence (GE Healthcare). Specific antibodies for lamin B1 (sc-6216), EGFP (sc-8334), Jagged1 (sc-8303), and Delta-1 (sc-9102) were obtained from Santa Cruz Biotechnology. Antibodies for Cre recombinase (ab24608) and NICD (07-1232) were purchased from Abcam and Millipore, respectively. The monoclonal antibody against Nrf2 used for immunoblotting was a gift from K. Itoh.

Oligonucleotide microarray

Total RNA was purified with the RNeasy Mini kit (Qiagen) after isolation with Trizol (Invitrogen) according to the manufacturer’s instructions. Murine Genome MOE 430A GeneChip arrays (Affymetrix), which contain probes for detecting 14,500 well-characterized genes and 4371 expressed sequence tags, were used to probe the gene expression in MEFs as described previously (12). Scanned output files were analyzed with Affymetrix GCOS (GeneChip Operating Software) and independently normalized to an average intensity of 500 before comparison.

Notch1 reporter constructs

The mouse Notch1 gene-regulatory region (−2 kb) from the major transcripts including the initiation codon sequence was isolated from C57BL/6J mouse liver genomic DNA by PCR. The region was directly cloned between Bgl II and Nco I sites in pGL3 basic (Promega) and confirmed by sequence analysis. In this construction, the ATG of Notch1 was replaced with the ATG of luciferase complementary DNA (cDNA). Serial promoter deletion fragments were prepared by the S1–Exo III nuclease reaction method followed by Nco I digestion. Then, the fragments were ligated to the site between Ecl 136II and Nco I sites of pGL3 basic. Serial point mutant and p-206 constructs were produced by PCR primer site-directed mutagenesis. Primers are shown in table S1. All constructs were confirmed by sequencing. Because pRLTK (Promega) bears an ARE sequence in the thymidine kinase promoter region, this ARE sequence was deleted to allow use of this vector for normalizing transfection efficiency. The improved normalizing vector, pRLTK-ΔARE, was constructed by deleting the Sma I and Pvu II fragment from pRLTK. Constructs used in transfection experiments were purified with plasmid kits (Qiagen) and detoxinized with an endotoxin removal kit (Mirus Bio Corp.).

Serial deletion reporter assay

MEFs or P19 cells were plated at a density of 2 × 105 per 60-mm dish 24 hours before DNA transfection. An equimolar concentration of the DNA of each construct (10 μg = 4.3 pmol of each reporter gene and 0.5 μg of pRLTK-ΔARE) was introduced into the cells by calcium phosphate coprecipitation (54). Transfections were repeated three to six times with independent plasmid DNA preparations. pBR322 plasmid DNA was used as a carrier to adjust the total amount of DNA. Cells were harvested 48 hours after transfection. Luciferase activity was measured according to the manufacturer’s instructions (Promega) and normalized to Renilla luciferase activity derived from pRLTK-ΔARE. Sulforaphane (LKT Labs) was used as an inducer of ARE-regulated genes (55). For transactivation assays, 4 μg of the reporter gene was transfected together with 8 μg of pCMVmNrf2 and 0.5 μg of pRLTK-ΔARE normalizing vector.

Isolation and purification of total RNA and RT-PCR

Cells were seeded at 60% confluence the day before treatment with vehicle or sulforaphane. Total RNA was prepared from MEFs or P19 cells with Trizol or Isogen (Wako), and then total RNA was purified with the RNeasy Mini kit. Mouse livers were perfused with ice-cold PBS before isolation of total RNA with the Versagene RNA purification system (Gentra Systems). RNA quality was confirmed by electrophoresis before reverse transcriptase reaction. Several genes were analyzed by SYBR Green real-time quantitative RT-PCR, and representative differentiation marker genes were analyzed by semiquantitative RT-PCR. cDNA was synthesized with the iScript system (Bio-Rad). Real-time PCR was performed on a Bio-Rad MyiQ real-time PCR machine with Applied Biosystems SYBR Green PCR master mix in triplicate 20-μl reaction volumes. The PCR efficiency was determined from a standard curve, and the Pfaffl method was used for calculation of fold changes (56). Melt curves and agarose gel electrophoresis were used to ensure specificity of the amplified product. Primers are shown in table S1. The intensity of bands was measured with ImageJ analysis software provided by the National Institutes of Health (NIH).

Analysis of DNA–nuclear factor binding

Nuclear extracts were prepared from MEF and P19 cells according to Dignam et al. (57). Protein concentrations of each nuclear extract were determined by the Bio-Rad protein assay with immunoglobulin G (IgG) as the standard. Probes were prepared by end-labeling 20 ng of the primary strand oligonucleotide with [γ-32P]adenosine triphosphate by T4 polynucleotide kinase, which was annealed subsequently to the complementary oligonucleotide. For the standard EMSA, a 25-μl reaction mixture containing ~2 × 104 cpm of probe nucleotide was incubated with 20 μg of protein of the nuclear extract in a buffer consisting of 10 mM Hepes (pH 7.9), 25 mM KCl, 1 mM EDTA, 1 mM ZnCl2, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 4 μg of poly(deoxyinosine-deoxycytidine), 0.05% NP-40, and 10% glycerol at 4°C for 30 min. For the competition experiments, each competitor oligonucleotide (table S1) was also added to the standard EMSA reaction mixture at a 25-fold molar excess to the probe, containing ~2 × 104 cpm, and the final volume was adjusted to 25 or 30 μl. The reaction products were loaded onto 5.0 or 8.0% polyacrylamide gels and run at 20 mA for 1.5 hours in 0.5× TBE buffer (45 mM tris-borate and 1 mM EDTA) at 4°C. All EMSA experiments were repeated three times, with nuclear extracts from MEF and P19 cells treated or untreated with 2.5 μM sulforaphane. Gels were dried and exposed to x-ray film at −80°C for 12 hours. In the case where recombinant Nrf2 and small MafK protein were used, the EMSA was performed as described previously (3).

ChIP assay

Formaldehyde cross-linking and chromatin fragmentation were performed as described previously (30). Diluted chromatin solution was incubated with an antibody to Nrf2 (H-300: sc-13032X, Santa Cruz Biotechnology), nonspecific IgG (normal rabbit IgG, sc-2027, Santa Cruz Biotechnology), or solution without antibody for 18 hours at 4°C with rotation. After washing and elution, precipitated DNA was dissolved in 60 μl of water. DNA solution (2 μl) as a template was used for PCR with the following primers: −187mNotch1-CP (5′-CTCCCTCCCGCGGCAGAGGCAC-3′) and −257mNotch1-CP (5′-CGCAGGAACCAGGGGCGGAGCC-3′). The primer sequences for promoters of β-Actin, which does not contain an ARE, and Gsta1, which bears functional AREs, have been described previously (17).

Statistical analysis

All values are expressed as mean ± SD except as noted otherwise. Statistical analysis was performed with unpaired Student’s t test or one-way analysis of variance (ANOVA) for comparison of multiple groups. Differences between groups were considered statistically significant when P < 0.05.


Acknowledgments: We thank H. Motohashi (Tohoku University), K. Itoh (Hirosaki University), S. Takahashi (University of Tsukuba), M. Morita (The Salk Institute for Biological Studies), A. P. McMahon (Harvard University), C. Murtaugh (University of Utah), I. Alcobia (University of Lisbon), and L. Parreira (University of Lisbon) for MafK expression vectors, antibody against Nrf2, pCaggs-nlsCre, pCaggs-Cre-EGFP, and pCAGGCre-ERTM expression vectors, genomic sequence information for RosaNICD mice, and human DLL1 and JAG1 expression plasmids, respectively. We also appreciate the technical assistance of P. Dolan, R. Rabold, P. Egner, J. Skoko, M. Iijima, and H. Sesaki. Funding: This work was supported by the Maryland Cigarette Restitution Fund; NIH grants R01 CA94076, R01 HL081205, P30 ES03819, T32 ES07141, and T32 CA009110; and Japan Science and Technology Corporation–Exploratory Research for Advanced Technology. S.S. is the recipient of a scholarship from the Samsung Foundation of Culture. Author contributions: N.W. designed and performed the research, established the NICD and compound genetically engineered mice, analyzed the data, and wrote the manuscript. S.S. conducted the ChIP assays. S.S. and J.W. conducted the RT-PCR analyses. S.L.S. established the conditional Keap1-disrupted MEFs. E.S.A. performed RT-PCR in MEFs and assisted in writing the manuscript. M.-K.K., V.M., and S.B. conducted and analyzed the initial microarray studies. M.Y. provided the Nrf2- and Keap1-null mice and helped evaluate the data. T.W.K. helped initiate the project and assisted with research design and manuscript preparation. Competing interests: There may be materials transfer agreements related to the new mouse constructs, per the policy of Johns Hopkins University.

Supplementary Materials

Fig. S1. Differential expression of Notch1 in Keap1-Nrf2 compound knockout MEFs and livers.

Fig. S2. Nqo1 and Hes1 gene expression after partial hepatectomies.

Table S1. Oligonucleotides used in experiments.

Table S2. PCR conditions for genotyping.


References and Notes

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