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Physiol. Genomics 32: 74-81, 2007. First published September 25, 2007; doi:10.1152/physiolgenomics.00126.2007
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Received 14 June 2007; accepted in final form 12 September 2007.
Physiological Genomics 32:74-81 (2007)
1094-8341/07 $8.00 © 2007 American Physiological Society

Genetic dissection of the Nrf2-dependent redox signaling-regulated transcriptional programs of cell proliferation and cytoprotection

Narsa M. Reddy1, Steven R. Kleeberger2, Masayuki Yamamoto3, Thomas W. Kensler1, Catherine Scollick1, Shyam Biswal1 and Sekhar P. Reddy1

1 Department of Environmental Health Sciences, The Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland
2 National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina
3 Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The beta zipper (bZip) transcription factor, nuclear factor erythroid 2, like 2 (Nrf2), acting via an antioxidant/electrophile response element, regulates the expression of several antioxidant enzymes and maintains cellular redox homeostasis. Nrf2 deficiency diminishes pulmonary expression of several antioxidant enzymes, rendering them highly susceptible to various mouse models of prooxidant-induced lung injury. We recently demonstrated that Nrf2 deficiency impairs primary cultured pulmonary epithelial cell proliferation and greatly enhances sensitivity to prooxidant-induced cell death. Glutathione (GSH) supplementation rescued cells from these defects associated with Nrf2 deficiency. To further delineate the mechanisms by which Nrf2, via redox signaling, regulates cellular protection and proliferation, we compared the global expression profiling of Nrf2-deficient cells with and without GSH supplementation. We found that GSH regulates the expression of various networks of transcriptional programs including 1) several antioxidant enzymes involved in cellular detoxification of reactive oxygen species and recycling of thiol status and 2) several growth factors, growth factor receptors, and integrins that are critical for cell growth and proliferation. We also found that Nrf2 deficiency enhances the expression levels of several genes encoding proinflammatory cytokines; however, GSH supplementation markedly suppressed their expression. Collectively, these findings uncover an important insight into the nature of genes regulated by Nrf2-dependent redox signaling through GSH that are involved in cellular detoxification and proliferation.

oxidative stress; antioxidants; lung; glutathione


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
AN IMBALANCE BETWEEN THE prooxidant load and the antioxidant defense system can contribute to or perpetuate the pathogenesis of many acute and chronic lung diseases, including malignancy (24). Several ex vivo and in vivo studies have clearly shown that the rapid induction of antioxidant enzyme expression in response to oxidant and toxic insults is mainly mediated by the antioxidant/electrophile response element (ARE or EpRE) commonly found in the regulatory regions (promoter and/or enhancers) of detoxifying and antioxidant enzymes (5, 20). Emerging evidence strongly supports a pivotal role for nuclear factor erythroid 2, like 2 (Nrf2), a beta zipper (bZip) transcription factor, in mediating the induction of several antioxidant enzymes in response to a variety of stimuli (5, 20). We have previously shown that Nrf2-deficient (Nrf2–/–) mice are more susceptible than wild-type (Nrf2+/+) mice to prooxidant stimuli such as hyperoxia (4), bleomycin (6), cigarette smoke (25), and LPS (29), suggesting that this transcription factor plays a key role in regulating prooxidant-induced lung inflammation, injury, and repair processes. Moreover, gene expression profiling in the lungs of Nrf2+/+ and Nrf2–/– mice exposed to the prooxidants listed above revealed that Nrf2 regulates hundreds of genes, including several antioxidant enzymes. The most notable of these genes are the glutathione (GSH) biosynthesis enzymes, GSH peroxidases (Gpxs), and GSH S-transferases (GSTs) (5, 20).

To better understand the mechanisms that confer pulmonary protection and are regulated by the Nrf2 transcription factor, we have utilized in vitro primary cultures isolated from the lungs of Nrf2+/+ and Nrf2–/– mice. We have focused on lung epithelial cells lining alveoli; these cells are constantly exposed to a prooxidant environment and play key roles in host defense, injury, and repair (3). Using this system, we have shown that an Nrf2 deficiency leads to defects in lung type II cell proliferation and greatly enhances sensitivity to H2O2-induced cell death, whereas GSH supplementation rescues cells from such defects by increasing the intracellular GSH levels (26), underscoring a prominent role of Nrf2-dependent GSH-induced signaling in type II cell proliferation and cellular protection against prooxidant stimuli. In this study, we demonstrate a significant insight into the network of transcriptional programs regulated by Nrf2-dependent GSH-induced signaling involved in epithelial cell proliferation and cellular protection against oxidant stress.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation and Culture of Murine Alveolar Type II Cells
The generation and characterization of mice with disruption of Nrf2 (Nrf2–/– mice) have been described elsewhere (17). Both wild-type (Nrf2+/+) and Nrf2–/– mice were maintained under the guidelines of the Institutional Animal Care and Use Committee of the Johns Hopkins University Bloomberg School of Public Health, and the animal protocols were approved by the Institutional Care and Use Committee. Type II alveolar epithelial cells were prepared from the lungs of these mice as described previously (8, 26). Briefly, lungs were perfused with 10 ml of 0.9% saline filled with 2 ml of dispase solution (0.8 U/ml), the trachea was tied, and the lungs were submerged in 1 ml of dispase solution and incubated at 37°C for 45 min. Lung tissue was gently teased and minced in a 100-mm culture dish containing 15 ml of HEPES-buffered DMEM and DNase I (100 U/ml). The cell suspension was passed through 100- and 40-µm cell strainers, centrifuged at 130 g for 8 min at 4°C, and incubated at 37°C on a 100-mm plate coated with mouse IgG for 2 h. After incubation, the nonadherent cells were collected by centrifugation and resuspended in HEPES-buffered DMEM supplemented with 10% FBS and keratinocyte growth factor (KGF) (10 ng/ml). The cells were plated on culture dishes coated with fibronectin and collagen, and medium was changed after the first day of culture and every 2 days thereafter. Cell viability of each preparation was determined by Trypan blue (0.1%) staining. Identification of type II epithelial cells was performed with Nile red as previously described (26). Type II cells isolated from Nrf2–/– mice were supplemented with 5 mM GSH (L-GSH; Sigma, G-6013). For GSH depletion, Nrf2+/+ cells were treated with 1 mM buthionine sulfoximine (BSO) for 6 and 24 h on day 5.

Expression Profiling
Total RNA was isolated from Nrf2+/+, Nrf2–/–, and Nrf2–/– supplemented with GSH (Nrf2–/–GSH) type II cell cultures from two independent samples (n = 2) on day 7 with the use of TRIzol (Invitrogen) followed by RNA cleanup with a Qiagen RNeasy kit. The isolated RNA was applied to mouse genome 430-2.0 arrays (Affymetrix, Santa Clara, CA), which contain probes for detecting ~45,000 well-characterized genes according to standard microarray protocol as previously described (13). Scanned output files were analyzed using Affymetrix GeneChip Operating Software and were independently normalized to an average intensity of 500. Further analysis was done, as described previously (13), by performing three pairwise comparisons for each group (Nrf2+/+ vs. Nrf2–/– and Nrf2–/–GSH vs. Nrf2–/–). To minimize the number of false positives, only those altered genes that showed a change ≥1.5-fold and appeared in all 6/9 comparisons were selected. In addition, the Mann-Whitney pairwise comparison test was performed to rank the results by concordance as an indication of the significance (P < 0.05) of each identified change in gene expression. Microarray data have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus database (accession no. GSE 7810; http://www.ncbi.nlm.nih.gov/geo/info).

Quantitative and Semiquantitative PCR Analyses
Total RNA (2 µg) was reverse transcribed using a superscript first-strand cDNA synthesis system (Invitrogen), and quantitative or semiquantitative PCR reactions were performed in triplicate. TaqMan gene expression assays for gpx2, gclc, gclm, gsta3, hmox, sod3, and actin were purchased from Applied Biosystems (Foster City, CA). The cycle threshold (CT) values for each gene were normalized to that of actin, and the relative value for the Nrf2–/– cells was set as one arbitrary unit (AU). For semiquantitative PCR analysis, gene-specific primers were used to amplify the cDNA products; cDNA was separated on agarose gel and stained with ethidium bromide. Values are shown as means ± SD, with n ≥ 3 for each experimental condition. Data were analyzed by a one-way ANOVA with Bonferroni correction. Significance in all cases was defined as P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
To further understand the cytoprotective mechanisms regulated by Nrf2-dependent GSH-induced signaling in lung epithelial cells, we have analyzed the gene expression profiles in Nrf2+/+ cells, Nrf2–/– cells, and Nrf2–/–GSH cells (Nrf2–/– cells supplemented with GSH) (see Fig. 1A). Our primary goal was to dissect the gene expression regulated by Nrf2 and Nrf2-dependent GSH-induced signaling. The genes that showed fold changes ≥1.7 were selected for further analysis. Genes whose expression in Nrf2–/–GSH cells is 75% or more of that in the Nrf2+/+ cells (100%) are considered fully recovered, i.e., GSH totally rescued their expression in the absence of Nrf2. Genes whose expression in Nrf2–/–GSH cells is 40–75% of that in Nrf2+/+ cells are considered partially rescued by GSH. The full list of genes that were commonly upregulated and downregulated by Nrf2 and Nrf2-GSH signaling are presented as Supplemental Materials (see Supplemental Tables S1 and S2, respectively; supplemental data are available at the online version of this article). Those genes whose expression in Nrf2–/–GSH cells was <30% are considered genes that may require Nrf2 for their expression. Because Nrf2-dependent GSH-induced signaling is critical for cell proliferation and protection against prooxidant stimuli, we compared the gene expression profiles of Nrf2+/+ and Nrf2–/–GSH cells with that of Nrf2–/– cells. The expression profiles that emerged after comparative analysis were further analyzed using Ingenuity Software. The expression profiles of genes that are involved in oxidative stress and cell proliferation can be found in Fig. 1 and in Tables 1 and Table 2, respectively. The resulting gene lists were divided into several categories (see below) to dissect out the Nrf2- and Nrf2-GSH-regulated transcriptional programs.


Figure 1
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  		Fig. 1. A: schematic representation of microarray experiment. WT, primary type II alveolar cells isolated from wild-type (Nrf2+/+) mice; KO, primary type II alveolar cells isolated from Nrf2-deficient (knockout; Nrf2–/–) mice. KO+GSH, Nrf2–/– cell culture supplemented with 5 µM glutathione (GSH; L-GSH reduced) for 5 days. B: list of antioxidant genes upregulated (open boxes) or downregulated (black boxes) in Nrf2+/+ cultures (left) and commonly upregulated (open boxes) or downregulated (black boxes) in Nrf2+/+ and Nrf2–/– cultures supplemented with GSH (right) compared with Nrf2–/– cultures. Gene symbols with their respective fold changes are represented in the boxes.

 

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  		Table 1. Commonly upregulated cell proliferation genes in WT and KOGSH cells

 

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  		Table 2. Commonly downregulated cell proliferation genes in WT and KOGSH cells

 
Gene Categories
Cytoprotective genes regulated by Nrf2 independently of GSH.
We categorized the genes that were overexpressed or underexpressed in Nrf2+/+ cells compared with Nrf2–/– cells and that did not return in Nrf2–/–GSH cells as Nrf2-regulated and GSH-independent genes (Fig. 1B, left). The upregulated genes include catalase, GST isoenzymes Gsta1, Gsta2, Gsta4, Gstm1 and Gsto2, glutamate-cysteine ligase, catalytic subunit (Gclc), glutamate-cysteine ligase, modifier subunit (Gclm) NAD(P)H dehydrogenase, quinone 1 (Nqo1), thioredoxin like 2 (Tnxl2), and thioredoxin reductase 1 (Txnrd1). All of these genes are previously reported to be regulated by Nrf2 (14, 15). We found that Nrf2 also downregulates several antioxidant genes in lung type II epithelial cells. They include nitric oxide synthase 2a (Nos2a), GSH reductase (GSR), prostaglandin D synthase (Ptgds), prostaglandin E synthase (Ptges), flavin-containing monooxygenase 3 (Fmo2), glyceraldehyde-3-phosphate dehydrogenase (Gapd), and superoxide dismutase 2 (Sod2). Some of the known targets of Nrf2, such as Gstm1, GST theta 1 (Gstt1), and Gclc, were also downregulated in Nrf2–/– cells, and GSH supplementation did not restore their expression.

Cytoprotective genes regulated by Nrf2 through GSH.
We identified antioxidant genes that were either upregulated or downregulated in the arrays of Nrf2+/+ and Nrf2–/–GSH cells compared with Nrf2–/– cells (Fig. 1B, right). The upregulated genes are GSH peroxidase 2 (Gpx2), Gsta3, aldehyde dehydrogenase 1A (Aldh1a1), and cytochrome b561 (Cyb561), and the fold differences for these genes when comparing Nrf2+/+ vs. Nrf2–/– and Nrf2–/–GSH vs. Nrf2–/– are as follows: Gsta3 (3.9 vs. 3.6), Aldh1a1 (5.9 vs. 2.1), and Cyb561 (2.1 vs. 3.2). Nrf2 also downregulated expression levels of several antioxidant enzymes such as superoxide dismutase 3 (Sod3), Gpx3, aldo-keto reductase 1b1 (Akr1b1), cytochrome 1b1 (Cyp1b1), cytochrome 7b1 (Cyp7b1), and monoamine oxidase B (Moab). In summary, these genes were upregulated in Nrf2–/– cells compared with Nrf2+/+ and Nrf2–/–GSH cells. The observed fold differences for downregulated genes are as follows: Sod3 (–10 vs. –24), Akr1b1 (–3.3 vs. –2.0), Cyp1b (1.53 vs. 2.45), Cyp7b1 (–5.9 vs. –11.4), and Moab (1.7 vs. 2.6) when comparing Nrf2+/+ vs. Nrf2–/– and Nrf2–/–GSH vs. Nrf2–/–.

Cytoprotective genes regulated by GSH in the absence of Nrf2.
These genes are differentially expressed in Nrf2–/–GSH cells only when compared with Nrf2–/– cells but are not present in wild-type cells. The expression of mitochondrial GSTs such as microsomal Gst2 (mGST2) and leukotriene C4 synthase (Ltc4s), which regulates LTC4 synthesis, was also upregulated by GSH. Several GST enzymes were downregulated by GSH, including Gstm2, Gstm3, Gstm5, and Gpx7 (data not shown).

Genes involved in cell proliferation regulated by Nrf2 through GSH.
We also identified several genes involved in cell proliferation that are either upregulated (Table 1) or downregulated (Table 2) in Nrf2+/+ and Nrf2–/–GSH cells compared with Nrf2–/– cells. We designated them as genes regulated by Nrf2 through GSH-dependent signaling. Forty-seven common upregulated genes involved in proliferation were overexpressed in wild-type cells compared with knockout cells and were induced with supplementation of GSH in knockout cell cultures. These genes were further categorized on the basis of function into growth factors, transmembrane receptors, kinases, transcription regulators, and other miscellaneous genes. Genes related to integrin signaling as well as transmembrane receptors involved in cell proliferation, migration, and adhesion were Nrf2-dependent and GSH signaling-inducible genes. The fold differences between Nrf2+/+ vs. Nrf2–/– and Nrf2–/–GSH vs. Nrf2–/– for some of these genes are as follows: Itga3 (9 vs. 11), Itga6 (2.9 vs. 3.1), Itgax (2.3 vs. 3.7), Itgb3 (1.1.8 vs. 1.7), Itgb6 (3.0 vs. 3.6), Ager (12.5 vs. 8.3), Cdh1 (2.4 vs. 3.5), Ceacam1 (1.9 vs. 2.7), Icam1 (1.6 vs. 2.0), Jup (2.0 vs. 2.1), Lama3 (2.0 vs. 2.8), Lama5 (1.8 vs. 2.0), Lamc2 (2.0 vs. 3.0), Mcam (3.6 vs. 3.0), and F3 (2.2 vs. 2.9). These are novel candidate genes that are regulated by Nrf2-induced signaling.

The mRNA expression levels of growth factors such as fibroblast factor 13 (Fgf13), heparin binding epidermal growth factor (Hbegf), neuregulin 1 (Nrg1), platelet-derived growth factor-β (Pdgfb), and transforming factor-β2 (Tgfb2) were significantly elevated in Nrf2+/+ and Nrf2–/–GSH cells. The fold differences between Nrf2+/+ vs. Nrf2–/– and Nrf2–/–GSH vs. Nrf2–/– were as follows: Fgf13 (7.0 vs. 8.5), Hbegf (3.0 vs. 2.6), Nrg1 (2.6 vs. 3.5), Pdgfb (2.8 vs. 3.2), and Tgfb2 (5.9 vs. 3.5). Nrf2 regulates kinases such as Dapk2, Prkci, and Epha2 through GSH signaling, which was not previously known. The fold changes for the above genes are 2.0 vs. 2.7, 1.7 vs. 1.9, and 2.1 vs. 2.4, respectively. Transcription factors regulated by Nrf2 through GSH signaling are Egr2, Foxa1, and Foxa2. These genes are upregulated in Nrf2+/+ and Nrf2–/–GSH cells by 2.0 and 3.0, 2.4 and 3.5, and 2.2 and 2.5, respectively, compared with Nrf2–/– cells (Table 1). Another category of genes regulated by Nrf2 through GSH is the transporters, including Mal (5.9 vs. 11), Mal2 (4.1 vs. 4.9), Slc6a14 (3.9 vs. 7.5), and Slc7a11 (3.7 vs. 2.2), whose fold differences are shown in parentheses.

Genes involved in cell proliferation downregulated by Nrf2 through GSH.
We also identified 48 downregulated genes that are classified as G protein-coupled receptors, growth factors, kinases, transcription regulators, transmembrane receptors, and other (Table 2). The G protein-coupled receptors that are downregulated through GSH are endothelial differentiation, lysophosphatidic acid G protein-coupled receptor 2 (Edg2) endothelial differentiation, sphingolipid G protein-coupled receptor 3 (Edg3), and neuropeptide Y receptor (Npy1r). The fold differences for Edg2, Edg3, and Npy1r were –2.2 vs. –4.7, –2.4 vs. –5.2, and –2.5 vs. –5.9, respectively. These genes are involved in the modulation of inflammatory responses by influencing cell migration and cell proliferation (1, 23). Integrin-{alpha}1 (Itga1) and integrin-{alpha}8 (Itga8) genes were downregulated in Nrf2+/+ and Nrf2–/–GSH cells. The fold changes for Itga1 and Itga8 were –1.9 vs. –2.5 and –3.7 vs. –3.7, respectively. Several transcription factors involved in DNA damage-dependent responses, such as Egr1 (–2.1 vs. –2.1), Maf (–2.0 vs. –2.7), Klf2 (–2.3 vs. –2.7), Pml (2.0 vs. 1.6), Prrx (1.5 vs. 10.7), Pttg (1.7 vs. 1.6), and Hif1{alpha} (3.9 vs. 1.7), were also downregulated by Nrf2 via GSH.

Nrf2 appears to downregulate the expression of several genes involved in negative regulation of cell proliferation. These genes include the following: collagen type XIV, {alpha}1 (Col14a1), chondritin sulfate proteoglycon 2 (Cspg2), decorin (Dcn), myosin light polypeptide chain (Mylk), phosphotidylethanolamine binding protein 1 (Pebp1), N-myc downstream-regulated gene 1 (Nrdg1), insulin-like growth factor binding protein 1 (Igfbp1), slit homolog 2 (Slit2), sprouty homolog 2 (Spry2), and tenascin (Tnc) through GSH. The fold differences for Col14a1 (1.9 vs. 1.6), Dcn (–2.0 vs. –7.3), Mylk (–6.9 vs. 21.6), Nrdg1 (–2.0 vs. –2.6), Igfbp1 (–5.4 vs. –3.1), and Tnc (–2.2 vs. –3.2) are shown in parentheses.

Inflammatory gene expression downregulated by Nrf2 through GSH.
Several inflammatory genes were downregulated in Nrf2+/+ and Nrf2–/–GSH cells compared with Nrf2–/– cells (Table 2). Some of the cytokines include the following: chemokine (C-C motif) ligand 2 (Ccl2), CCL7, CCL11, chemokine (C-X-C) ligand 10 (Cxcl10), Cxcl12 (also known as stromal-derived factor 2), and Cxcl14. The fold changes for Ccl2 (–1.6 vs. –1.7), Ccl7 (–2.1 vs. –2.5), Ccl11 (17.6 vs. 26.9), Cxcl5 (–2.8 vs. –2.1), Cxcl10 (–2.7 vs. –3.2), Cxcl12 (–2.0 vs. –9.2), and Cxcl14 (–3.7 vs. –2.2) in Nrf2+/+ vs. Nrf2–/– and Nrf2–/–GSH vs. Nrf2–/– are shown in parenthesis. Other inflammatory molecules that were downregulated through GSH include transcription factor CCAAT enhancer binding protein-{delta} (Cebp{delta}), pentraxin 3 (Ptx3), serpina3n, and interleukin 6 signal transducer (Il6st), and the fold differences are as follows: Cebp{delta}, –1.7 vs. –2.0; Ptx3, –1.9 vs. –8.3; serpina3n, –4.9 vs. 5.1; and Il6st, –1.5 vs. –3.2.

Validation of Microarray Data
We have performed quantitative and semiquantitative PCR using gene-specific primers (see Supplemental Table S3) for validation of gene expression profiles. Seventeen genes were selected as targets from the Nrf2-regulated and GSH-independent gene list and Nrf2-regulated genes dependent on GSH (Fig. 2). RNA expression for all six genes (gclc, gclm, gsta3, gsta4, gsta1, and gsta2) selected from the Nrf2-regulated and GSH-independent gene list varied markedly between Nrf2+/+ and Nrf2–/– cells and did not return in Nrf2–/–GSH cells. These results confirmed the expression patterns of the microarrays. Similarly, quantitative or semiquantitative PCR data for 10 genes selected from the Nrf2-regulated and GSH-dependent gene list confirmed the expression patterns of the microarray data, i.e., expression of these genes returned in Nrf2–/–GSH cells.


Figure 2
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  		Fig. 2. Validation of microarray data. Preparation of RNA samples and cDNA was performed as described in MATERIALS AND METHODS. A: expression levels of Gclc, Gclm, Gsta3, Hmox1, and Sod3 in WT (Nrf2+/+), KO (Nrf2–/–), and KO+GSH (Nrf2–/–GSH) RNA samples as determined by quantitative PCR using TaqMan gene expression assays. Cycle threshold values for each gene were normalized with that of actin. B: semiquantitative RT-PCR analysis of genes. Left: genes whose expression is low in KO cells compared with WT cells. Right: genes whose expression is commonly up- or downregulated in WT and KO+GSH cell cultures compared with KO group. β-Actin (actin b) gene expression was used as reference. C: quantitative and semiquantitative PCR analysis of commonly up- or downregulated cell genes in WT, KO, and KO+GSH cells. D: a simplified scheme of Nrf2-mediated induction of GSH-synthesizing enzymes and their downstream effector functions in response to cellular stress. Under conditions of stress, Nrf2 accumulates in the nucleus and on dimerization with the other bZip transcription factors, such as MAF, JUN, or ATF family of proteins; Nrf2 binds to the ARE sequence and drives the transcription. Open circle with X represents the MAF, JUN, or ATF protein.

 
GSH Depletion Greatly Diminishes the Expression Levels of Antioxidant Genes
To determine whether depletion of GSH would have an effect on antioxidant gene expression, Nrf2 cells were treated with BSO for 6 and 24 h, RNA was isolated, and quantitative and semiquantitative PCR analyses were used to analyze the expression levels of several antioxidant genes. The mRNA expression levels of several antioxidant enzymes, such as gclc, gclm, gsta3, gsta4, gsta1, and gsta2, were markedly lower in Nrf2+/+ cells treated with BSO as early as 6 h and remained low at 24 h compared with vehicle-treated control group (see Supplemental Fig. E1).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We and others have shown by expression profiling that Nrf2 regulates the expression of numerous genes involved in various biological processes; in particular, the induction of several antioxidant enzymes is a salient feature (5, 20). Using primary cell cultures of Nrf2-sufficient and Nrf2-deficient cells with and without exogenous GSH supplementation, we have deciphered transcriptional programs that are induced by Nrf2 via GSH-induced signaling (see Supplemental Tables S1 and S2 for full gene lists). Consistent with previous reports, we found a lower level of expression of several antioxidant enzymes and cytoprotective genes in Nrf2–/– cells than in Nrf2+/+ cells. Interestingly, supplementation of GSH to Nrf2–/– cells restored the expression of several antioxidant enzymes to the levels present in Nrf2+/+ cells (Fig. 1B; see Supplemental Table S1 for more details), suggesting that Nrf2 regulates gene expression in part via GSH-induced signaling. Moreover, we found that the expression of several antioxidant enzymes is negatively regulated by GSH-dependent redox state. For instance, we found elevated levels of Sod3, Gpx3, and Gstt1 in Nrf2–/– cells (Fig. 1B), which exhibit high levels of reactive oxygen species (ROS). Sod3 is known to scavenge the superoxide radicals, whereas Gstt1 and Gpx3 play key roles in the detoxification of electrophilic compounds. It is likely that these genes are induced in Nrf2–/– cells and, in the absence of GSH, probably counteract the oxidative stress exerted by cellular metabolism. In the case of Nrf2+/+ cells and Nrf2–/–GSH cells, where GSH levels are higher than for Nrf2–/– cells, a feedback loop of GSH may negatively regulate Sod3, Gpx3, and Gstt1 expression by an unknown mechanism in the absence of oxidative stress.

We found that Nrf2-GSH-dependent signaling regulates the expression of several genes involved in cell proliferation, consisting of various receptors, growth factors, kinases, and transcription regulators (Table 1). Some of the receptors include the following: Epha2, Ager1, Icam1, and F3 and integrins (Itga3, Itgb6, and Itgax) that are regulated by Nrf2 via GSH. Both integrins and Epha2 tyrosine kinase receptor activation lead to activation of a wide range of cellular proteins, including MAP kinases, which subsequently phosphorylate various effector transcription factors that regulate gene expression (19). Ager1 has been shown to suppress oxidant stress via epidermal growth factor receptor-activated signaling (2).

Nrf2-GSH signaling also upregulates the expression of several growth factors, such as Nrg1, Ntf5, Hbegf, Tgfβ2, Pdgf-B, VWF, and Fgf13. Nrg1 and Hbegf bind to the ErbB receptor and initiate signal transduction pathways that regulate cell proliferation during development and in response to injury (11). Prkci, a serine and threonine kinase (15), and Epha2 (18) activate MAP kinase pathways, thereby regulating gene expression. GSH also upregulates the expression of various transcription factors such as Foxa1 and Foxa2, which play key roles in lung development and function (31). Our data (Table 1) suggest that Nrf2-GSH signaling regulates the expression of several cell structural proteins such as laminins (lama3 and lama5), occludins (Ocln), junction proteins such as plakoglobin (Jup), microtubule-associated protein 7 (MAP7), and cadherens such as Cdh1. All of the above genes are required for cell proliferation, migration, and adhesion (10, 22).

Interestingly, our data (Table 2) suggest that Nrf2-regulated and GSH-dependent signaling downregulates the expression of several chemokines (e.g., Ccl2, CcL7, CcL8, CcL11, CxcL10, Cxcl12, and Cxcl14). The suppression of several chemokines by GSH in our experimental conditions suggests that Nrf2-dependent GSH-induced redox signaling may control inflammatory responses (14). Consistent with this notion, we have shown previously that Nrf2 deficiency enhances the expression levels of several proinflammatory cytokines including chemokines (29). Other inflammatory molecules that are downregulated through GSH are Cebp{delta} transcription factor, Il6st, Ptx3, and Serpina3 (Table 2). Cebp{delta} activates the expression of cyclooxygenase 1 and 2 (28). Ptx3 regulates cytokines such as Cxcl6, Ccl13, TNF{alpha}, and Il1β. Serpin A family proteins are required for the release of leukocyte proteases during the inflammatory response (16). Liposaccharide binding protein is known to play a role in LPS-dependent monocyte functions. Although the exact mechanisms by which GSH controls the regulation of the expression of the above genes remain to be investigated, recent studies have shown that protein glutathionylation and deglutathionylation affect the functions of various proteins, including transcription factors such as NF-{kappa}B, IkkB, and c-Jun (7, 13, 21, 27). It is likely that decreased GSH levels in Nrf2–/– cells may cause dysregulation of signal transduction pathways and the effector transcription factor activation essential for gene expression. For example, we have shown that Nrf2 deficiency enhances IkkB phosphorylation and NF-{kappa}B DNA binding activity. Depletion of GSH in wild-type cells with BSO resulted in downregulation of several Nrf2-dependent genes (Supplemental Fig. E1), further confirming that Nrf2-dependent redox signaling through GSH is required for gene expression. Additional studies delineating the glutathionylation and deglutathionylation mechanisms regulated by Nrf2-GSH-induced signaling may provide further insight into the roles of redox signaling in type II cell proliferation and differentiation during lung injury and repair. Although the factors that regulate the induction of several cytoprotective genes in the absence of Nrf2 are unclear, it is likely that other transcription factors such as Jun-D or c-Myc may compensate to upregulate the expression of genes involved in cytoprotection and cell proliferation, and this is likely mediated by TPA response element, or TRE (which is often embedded in ARE site), or E-box response element, respectively. Consistent with this view, it has been shown that Jun-D and Myc transcription factors regulate the expression of several genes involved in cytoprotection (9, 12, 30).

In summary, our data obtained with a primary epithelial cell culture system uncovered for the first time that Nrf2 uses GSH as an effector to regulate the expression of several genes (including novel targets) involved in cell proliferation and cellular protection against oxidants (see Fig. 2C). Intriguingly, we found that GSH differentially regulates the expression of several genes in the absence of Nrf2, suggesting an autoregulatory loop between Nrf2- and Nrf2-GSH-regulated transcriptional programs, probably to maintain cellular homeostasis.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants HL-66109 and SCCOR-P50-HL-073994 (to S. P. Reddy), HL-81205 (to S. Biswal), CA-94076 (to T. W. Kensler), and P30-ES-038819.


   ACKNOWLEDGMENTS
 
We thank Hannah Lee for assistance in microarray analysis.


   FOOTNOTES
 
Address for reprint requests and other correspondence: N. M. Reddy, The Johns Hopkins Bloomberg School of Public Health, Dept. of Environmental Health Sciences, Rm. E7547, 615 North Wolfe St., Baltimore, MD 21205 (e-mail: sreddy{at}jhsph.edu).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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