Genetic disruption of the Nrf2 compromises cell-cycle progression by ...

NIH Public Access Author Manuscript Oncogene. Author manuscript; available in PMC 2009 February 23.

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Published in final edited form as: Oncogene. 2008 October 2; 27(44): 5821–5832. doi:10.1038/onc.2008.188.

Genetic disruption of the Nrf2 compromises cell-cycle progression by impairing GSH-induced redox signaling NM Reddy1, SR Kleeberger2, JH Bream3, PG Fallon3, TW Kensler1, M Yamamoto4, and SP Reddy1 1Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA 2National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA 3Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA


4Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai, Japan

Abstract Genetic disruption of Nrf2 greatly enhances susceptibility to prooxidant- and carcinogen-induced experimental models of various human disorders; but the mechanisms by which this transcription factor confers protection are unclear. Using Nrf2-proficient (Nrf2+/+) and Nrf2-deficient (Nrf2−/−) primary epithelial cultures as a model, we now show that Nrf2 deficiency leads to oxidative stress and DNA lesions, accompanied by impairment of cell-cycle progression, mainly G2/M-phase arrest. Both N-acetylcysteine and glutathione (GSH) supplementation ablated the DNA lesions and DNA damage–response pathways in Nrf2−/− cells; however only GSH could rescue the impaired colocalization of mitosis-promoting factors and the growth arrest. Akt activation was deregulated in Nrf2−/− cells, but GSH supplementation restored it. Inhibition of Akt signaling greatly diminished the GSH-induced Nrf2−/− cell proliferation and wild-type cell proliferation. GSH depletion impaired Akt signaling and mitosis-promoting factor colocalization in Nrf2+/+ cells. Collectively, our findings uncover novel functions for Nrf2 in regulating oxidative stress-induced cell-cycle arrest, especially G2/M-checkpoint arrest, and proliferation, and GSH-regulated redox signaling and Akt are required for this process.


Keywords oxidative stress; Nrf2; cell cycle; G2/M-checkpoint; Akt

Introduction Cell proliferation following tissue injury caused by prooxidant and toxicant exposure is critical for maintaining tissue homeostasis. However, dysregulation of cell-cycle progression as a result of oxidative stress can lead to the development of various diseases, including malignancy (Golubnitschaja, 2007). However, the mechanisms involved in these processes are not yet completely understood. Antioxidant proteins/peptides (for example, superoxide dismutases,

Correspondence: Dr SP Reddy, Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Room E7610, 615 North Wolfe Street, Baltimore, MD 21205, USA. E-mail: [email protected]. Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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catalase, glutathione peroxidases, peroxiredoxins, thioredoxins, glutaredoxins and glutathione (GSH)) maintain the ‘reducing’ environment of the cell by inactivating endogenous reactive oxygen species (ROS) and preventing ROS-initiated reactions induced by external insults. An imbalance between prooxidant load and the endogenous cellular antioxidant defense system can lead to oxidative stress (Valko et al., 2007). Thus, delineating the factors that regulate cellular redox status and their downstream effector functions is of considerable significance. One of the critical regulators of cellular redox status is the Nrf2 transcription factor, a cap ‘n’ collar basic leucine zipper protein. Nrf2 is required for constitutive and inducible expression of Gclm and Gclc, which are required for GSH biosynthesis. Nrf2 also regulates expression of other cellular detoxifying enzymes, such as NQO1, HO-1 and glutathione S-transferases (Nguyen et al., 2003). Studies using Nrf2-deficient (Nrf2−/−) mice have revealed protective roles for this transcription factor in various models of human disorders of the lung, liver, kidney, brain, neuron, skin and circulation (see recent reviews Cho et al., 2006; Kensler et al., 2006). In general, Nrf2−/− mice are more susceptible than wild-type mice to various experimental models of diseases linked to inflammation, injury and repair processes. 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 cellular detoxifying enzymes is a salient feature (Cho et al., 2006; Kensler et al., 2006).


We and others have shown that a deficiency of Nrf2 augments lung injury caused by various prooxidants, such as butylated hydroxytoluene (Chan and Kan, 1999), hyperoxia (Cho et al., 2002), diesel exhaust particles (Aoki et al., 2001; Li et al., 2004), cigarette smoke (Rangasamy et al., 2004) and lipopolysaccharide (Thimmulappa et al., 2006). Alveolar epithelial cells are critical for maintaining lung tissue homeostasis and are also important in host defense and innate immunity (Qian et al., 2006; Sugahara et al., 2006; Serrano-Mollar et al., 2007). We have recently demonstrated that disruption of Nrf2 leads to alveolar epithelial cell growth arrest accompanied by oxidative stress (diminished levels of GSH and increased levels of ROS; Reddy et al., 2007a), which is correlated with diminished mRNA expression levels of genes encoding Gclc and Gclm enzymes and dysregulation of networks of transcriptional programs involved in cell proliferation (Reddy et al., 2007b). GSH supplementation rescued Nrf2−/− cell growth arrest and restored gene expression involved in cell proliferation suggesting a critical role for Nrf2-regulated GSH-induced redox signaling in epithelial cell proliferation. In the present study, we have used primary alveolar epithelial cultures isolated from Nrf2+/+ and Nrf2−/− mice to investigate the mechanisms contributing to the reversible growth arrest caused by Nrf2 deficiency. Our findings for the first time provide genetic and pharmacological evidence that supports a critical role for Nrf2-dependent, GSH-induced redox signaling in regulating G2/M-checkpoint control and epithelial cell-cycle progression. We have further demonstrated that Akt signaling is important in this process.


Results A deficiency of Nrf2 induces G2/M-checkpoint arrest and DNA lesions, but GSH supplementation rescues these defects To define the mechanisms contributing to the reversible cell-cycle arrest caused by Nrf2 deficiency, we first analysed the cell-cycle distribution in Nrf2+/+, Nrf2−/− and Nrf2−/−GSH cells. Fluorescence-activated cell sorting (FACS) analysis revealed that the percentage of the cells in the G1 phase was very similar in Nrf2+/+, Nrf2−/− and Nrf2−/−GSH cells (Figure 1a), but the percentage of cells in S phase was significantly higher in Nrf2+/+ (25%) and Nrf2−/−GSH cells (28%) than in Nrf2−/− cells (7.5%). In contrast, the percentage of Nrf2−/− cells in G2 phase (29.5%) was markedly higher than that of Nrf2+/+ (6.7%) and Nrf2−/−GSH (7%) cells. This reduction in S phase and significant increase in G2-phase distribution in Nrf2−/− cells indicate that Nrf2 deficiency induces G2/M-checkpoint arrest. We have recently Oncogene. Author manuscript; available in PMC 2009 February 23.

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shown that Nrf2−/− cells, which are viable and display differentiated phenotypes, have elevated levels of ROS that are correlated with diminished levels of GSH (Reddy et al., 2007a). Given the known genotoxic effects of ROS, we examined the DNA lesions produced in Nrf2−/− cells to determine the relative genomic integrity of these cells. To test for DNA damage, we used fluorescein isothiocyanate (FITC)-conjugated avidin to detect 8-oxodeoxiguanosine, an oxidative DNA lesion. Nrf2−/− cells showed increased FITC–avidin staining (Figure 1b), whereas both Nrf2−/− cells treated with GSH and wild-type cells had no apparent FITC–avidin staining in the nucleus (Figure 1b). To confirm that DNA damage had indeed occurred, we performed TdT-mediated dUTP nick end labeling (TUNEL) staining of these cells. We found that 95% of the Nrf2−/− cells were TUNEL-positive, whereas none of the wild-type cells or GSH-treated Nrf2−/− cells (Nrf2−/−GSH) were TUNEL-positive (Figure 1c). Consistent with this result, depletion of GSH from wild-type cells using buthionine sulfoximine- (BSO) enhanced oxidative stress and decreased cell proliferation, although supplementation of GSH rescued this inhibition (data not shown). Collectively, these results strongly suggest that Nrf2 deficiency leads to G2/M-phase arrest resulting from a lack of sufficient intracellular GSH (Reddy et al., 2007a). Oxidative stress induces the ATM pathway and its effectors in Nrf2−/− cells

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The accumulation of genetic damage induces various signaling cascades and the activation of ATM is a hallmark of such damage (Kastan and Lim, 2000). To assess the involvement of ATM-mediated signal transduction events in Nrf2−/− cells, we conducted the immunostaining experiments using antibodies recognizing phospho-specific ATM, p53, Rb, Chk1, Chk2 or Cdc25c (Figure 2). We chose these particular proteins because they are known to regulate cellcycle arrest. A high level of ATM phosphorylation was detected in Nrf2−/− cells as indicated by immunostaining using anti-phospho-serine (Ser1981) antibody (Figure 2a). Immunostaining with an anti-phospho-p53 antibody (Ser15) revealed a robust phosphorylation of p53 in Nrf2−/− cell, a very weak phosphorylation in Nrf2−/−GSH cells and no phosphorylation in Nrf2+/+ cells (Figure 2b). Phosphorylation of Rb (Ser807/811) was decreased in Nrf2−/− cells, and supplementation with GSH enhanced the Rb phosphorylation, although it was not completely restored to the level found in Nrf2+/+ cells (Figure 2c). Chk1 and Chk2 kinases inactivate Cdc25c by phosphorylating Ser216, thereby generating a consensus binding site for 14-3-3 proteins (Peng et al., 1997; Chen et al., 2003), which leads to the sequestration of this phosphatase into the cytoplasm, and an inhibition of Cdk1 activity (Lopez-Girona et al., 1999). We therefore assessed the phosphorylation status of Chk1, Chk2 and Cdc25c. Nrf2−/− cells were positively stained with the antibodies detecting phospho-specific Chk1 (Ser345; Figure 2d) and Chk2 (Thr68; Figure 2e), indicating the activation of these kinases. Little or no immunofluorescence was observed in either Nrf2+/+ cells or Nrf2−/−GSH cells after staining with these antibodies. Immunostaining with anti-phospho-Cdc25c (Ser216) antibody revealed that Cdc25c was inactivated in Nrf2−/− cells, but GSH supplementation was able to decrease the Ser216 phosphorylation to undetectable levels, similar to the situation seen in wild-type cells (Figure 2f). Nrf2 deficiency inhibits the colocalization of mitosis-promoting factor complex proteins The transition from G2 phase to mitosis is triggered by the Cdc25-mediated activation (dephosphorylation) of the cyclin B1/Cdk1 complex known as the MPF complex. Cyclin B1/ Cdk1 is activated by phosphorylation of Thr160 (Thr161 in mice) and the dephosphorylation of Thr14 and Tyr15 (Tyr18 in mice). As shown in Figure 3a, immunostaining with antibodies recognizing phospho-Cdk1 (Thr161; active) and cyclin B1 revealed a colocalization of these two proteins in the nuclei of Nrf2+/+ cells (top panel). In contrast, these proteins were not colocalized in Nrf2−/− cells (middle panel). GSH supplementation of Nrf2−/− cells restored the colocalization of these proteins, producing a pattern resembling that in wild-type cells (bottom panel). Co-immunocolocalization studies using antibodies specific for inactive pCdk1 (Tyr18) Oncogene. Author manuscript; available in PMC 2009 February 23.

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and cyclin B1 revealed that inactive Cdk1 was localized to the plasma membrane, whereas cyclin B1 was present in both the cytoplasm and nucleus of Nrf2+/+ cells (Figure 3b, top panel). In contrast, inactive pCdk1 and cyclin B1 were both colocalized to the cytoplasm and nucleus in Nrf2−/− cells (middle panel). However, supplementation with GSH restored the localization of inactive pCdk1 to the plasma membrane in Nrf2−/− cells, generating a pattern similar to that in wild-type cells (bottom panel). Collectively, these results indicate that redox signaling dependent on Nrf2–GSH is critical for the proper colocalization of the MPF proteins, cyclin B1 and Cdk1, in the nucleus. The antioxidant N-acetylcysteine abolishes DNA lesions but cannot restore cell proliferation


To determine whether scavenging ROS alone was sufficient to induce Nrf2−/− cell proliferation, we supplemented primary cell cultures from day 1 with N-acetylcysteine (NAC), a precursor form of GSH. As expected, NAC completely eliminated the elevated levels of ROS and DNA lesions in Nrf2−/− cells (Figure 4a); however it failed to induce the cell proliferation of Nrf2−/− cells. We next examined the effects of NAC on the activation status of DNA damage–response pathways. NAC supplementation completely abolished the activation of ATM, p53, Chk1, Chk2 and Cdc25c proteins in Nrf2−/− cells (Figure 4b). In contrast, it failed to restore Cdk1 phosphorylation or the colocalization of cyclin B1/Cdk1 proteins in the cellular compartments to the pattern seen in wild-type and Nrf2−/−GSH cells (Figure 4c). These results suggest that ROS scavenging is not sufficient to induce G2/M-phase progression. The lack of an effect of NAC on Nrf2−/− cells is attributed to diminished levels of Gclc and Gclm enzymes (Reddy et al., 2007a), which are critical for GSH biosynthesis and the conversion of NAC to GSH. To verify the results of immunofluorescence studies, we have analysed the phosphorylation status of several cell-cycle proteins in the Nrf2+/+, Nrf2−/−, Nrf2−/−GSH and Nrf2−/−NAC cell lysates by western blot analysis (Figure 5). Immunoblot analysis of Nrf2+/+, Nrf2−/−, Nrf2−/−GSH and Nrf2−/−NAC cell lysates with phospho-Rb (Ser807/811) antibody and phosphoCdc25c (Ser216) has shown the phosphorylation status of these proteins similar to that of immunofluorescence analysis (Figures 3 and 4). Analysis for phospho-Cdk1 (Thr161) revealed greatly diminished levels of phosphorylation in Nrf2−/− cells as compared to Nrf2+/+ and Nrf2−/−GSH cells; however, in Nrf2−/− cells NAC failed to restore Cdk1 phosphorylation to levels comparable to those seen in Nrf2+/+ and Nrf2−/−GSH cells. An Nrf2 deficiency impairs Akt signaling


We reasoned that decreased GSH levels in Nrf2−/− cells might cause a dysregulation of signal transduction pathways and the effector transcription factor activation required for the gene expression that is involved in cell-cycle progression and proliferation. This notion is based on recent studies that have suggested a role for protein glutathionylation, a reversible posttranslational mechanism in modulating the activities of the redox-sensitive thiol proteins, especially those are involved in signal transduction and cell proliferation (Dalle-Donne et al., 2007a). In the present study, we mainly examined the role of ERK1/2, Stat3 and Akt kinases, which are known to be important in cell proliferation. Both wild-type and Nrf2−/−GSH cells were incubated without or with the PI3K inhibitor LY294002, the Akt-specific inhibitor AktII, the ERK inhibitor UO126 or Stat3 inhibitor AG490 (Figure 6a). The PI3K and Akt inhibitors had a dramatic inhibitory effect on the proliferation of both Nrf2+/+ and Nrf2−/−GSH cells. In contrast, the ERK and Stat3 inhibitors had no significant effect, indicating that Akt-mediated signaling is critical for GSH-induced Nrf2−/− cell proliferation. To further define the molecular basis of these observations, we examined the activation status of ERK1/2, Stat3 and Akt kinases in response to growth factor stimuli. Immunoblot analysis revealed that decreased levels of platelet-derived growth factor (PDGF) and insulin-induced Akt, ERK1/2 and Stat3 kinase phosphorylation in Nrf2−/− cells when compared to Nrf2+/+ cells (Figure 6b). The

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phosphorylation and expression levels of Akt, ERK and Stat3 kinases were restored when the Nrf2−/− cell cultures were supplemented with GSH. Real-time PCR analysis revealed no significant changes in the expression levels of Akt isoforms (Figure 6c) suggesting that an Nrf2 deficiency may not affect the Akt levels at the transcriptional level. GSH depletion is associated with impairment of cell-cycle progression, a dysfunctional Akt signaling and lack of colocalization of MPF complex proteins in wild-type cells


To confirm the role of GSH signaling in cell-cycle progression, we depleted GSH in Nrf2+/+ cells by treating the cells with 0.5 mM BSO on day 4 for 24 h, then analysing the cell-cycle distribution, and activation of Akt and MPF in the treated cells. The FACS analysis revealed that 85% of the BSO-treated Nrf2+/+ cells were in G1 phase, as compared to 58% in untreated cells; thus most of the cells were arrested at the G1/S transition. However, the percentage of cells that were in S or G2 phase was significantly reduced after GSH depletion (Figures 7a and b). We then examined the phosphorylation and protein levels of Akt and ERK1/2 kinases by immunoblotting. GSH depletion decreased the phosphorylation status as well as the protein levels of Akt, whereas BSO treatment significantly reduced the phosphorylation, but not the level of expression, of ERK1/2 kinases (Figure 7c). Coimmunostaining with phospho-Cdk1 (Thr161; active) and cyclin B1, revealed that GSH depletion inhibited both the Cdk1 phosphorylation at Thr161 and association with cyclin B1. In contrast, BSO treatment inhibited the dephosphorylation of Cdk1 at Tyr18 and led to an association of inactive phospho-Cdk1 (Tyr18) with cyclin B1 in the cytoplasm and nucleus. However, inactive phospho-Cdk1 (Tyr18) was localized to the cell membrane in untreated cells (Figure 7d).

Discussion


In the present study, we have for the first time provided the genetic and pharmacologic evidence that Nrf2-dependent GSH-induced redox signaling is critically required for cell-cycle progression and cell proliferation. Our data demonstrate that genetic disruption of Nrf2 leads to growth arrest, particularly in the G2/M phase with relatively very low numbers of cells in S phase. Of particular note is the fact that we have demonstrated that inhibiting the ROS and DNA damage–response pathways alone is not sufficient to induce cell proliferation and additional signaling induced by GSH is also critical for cell-cycle progression (Figure 8). The inability of NAC to restore the defect in Nrf2−/− cell proliferation is consistent with our previous observation that Nrf2 deficiency diminishes the expression levels of Gclc and Gclm (component of γ-GCS; Reddy et al., 2007a), whose activity is essential for the biosynthesis of GSH from its precursor NAC. It is noteworthy that Nrf2 deficiency appears to be more specific to alveolar epithelial cells. We found that renal epithelial cells isolated from Nrf2−/− mice could proliferate, although at slower rate, and these cells showed less oxidative stress, as evidenced by DCF staining (Supplementary Figure S5). Various signal transduction pathways are rapidly activated in response to the oxidative stress caused by DNA damaging agents (Su, 2006) and the activation of ATM is crucial for the initiation of signaling pathways that block cell-cycle progression at the G1/S and G2/M interfaces (Bakkenist and Kastan, 2003;Helt et al., 2005). Immunostaining has revealed high levels of ATM phosphorylation and its downstream effectors, p53 (Meulmeester et al., 2005), and Chk1 and Chk2 kinases (Niida and Nakanishi, 2006) in Nrf2−/− cells, when compared to wild-type and Nrf2−/−GSH cells (Figure 2). By inhibiting cyclin B1/Cdk1 activity (Taylor and Stark, 2001) and cyclin A1/Cdk2-mediated phosphorylation of the Rb protein, p53 maintains G2/M-phase arrest (Timmers et al., 2007). Chk1 and Chk2 kinases inactivate Cdc25c by phosphorylating Ser216 and this phosphorylation generates a consensus binding site for 14-3-3 proteins (Peng et al., 1997), leading to sequestration of this phosphatase in the cytoplasm. The inactivation of Cdc25c by Chk1 and Chk2 kinases inhibits cyclin B1/Cdk1


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activity and blocks the G2/M-phase transition. Consistent with these results, our coimmunocolocalization studies revealed inactive pCdk1 (phospho-Tyr18) and cyclin B1 in the cytoplasm and in nuclei of Nrf2−/− cells (Figure 3b). In contrast, inactive Cdk1 was localized to the cell membrane in Nrf2+/+ cells. GSH supplementation restored inactive Cdk1 on the cell membrane of Nrf2−/− cells, producing an expression pattern similar to that seen in wild-type cells (Figure 3b). We did not observe any significant difference in the localization of cyclin B1, which was found both in the cytoplasm and in the nucleus. In analogous experiments, we found a lack of colocalization of active Cdk1 (Thr161) and cyclin B1 in Nrf2−/− cells, in contrast to their localization in the nucleus in both Nrf2+/+ and Nrf2−/−GSH cells. In agreement with these results, depletion of GSH in wild-type cells caused a similar phenomenon (Figure 7d). Taken together, these data suggest that oxidative stress impairs the colocalization of MPF, thereby leading to G2/M-phase arrest. Interestingly, we found that although supplementation with NAC was able to ablate the DNA lesions and DNA damage–response pathways activated by ATM (Figure 4b), it failed to restore proper colocalization of the MPF complex proteins, Cdk1 and cyclin B1 (Figure 4c) in a manner similar to that produced by GSH supplementation (Figure 3). These results suggest that the inability of NAC to induce the proliferation of Nrf2−/− cells may be due in part to a mislocalization of MPF complex proteins, whose activity is necessary for G/M-phase progression (Nurse, 1990;Nigg, 2001).


Our findings indicate that diminished levels of GSH, resulting from Nrf2 deficiency, can lead to dysregulation of various signal transduction pathways, thereby affecting effector transcription factor activation and gene expression. The deregulation of growth factor induced ERK1/2, Stat3 and Akt kinase signaling that we observed in Nrf2−/− cells, but not in Nrf2−/−GSH cells (Figure 6b), suggests that Nrf2-regulated GSH levels are critical for the activation of these kinases. Akt and ERK signaling are known to regulate cell-cycle progression. For example, activated ERK1/2 induces the transcription of cyclin D1 (Yoshida et al., 2003), which associates with Cdk4 and Cdk6 and leads to the phosphorylation of the Rb protein (Bracken et al., 2004) and stabilization of c-Myc (Sears et al., 2000). The transcription factor c-Myc regulates cell-cycle progression in turn by suppressing the expression of cellcycle/growth arrest genes (Gartel and Shchors, 2003). Akt-induced signaling modulates the activation of Cdk1 at the G2/M-phase transition. On the other hand, Akt phosphorylates Chk1 kinase at Ser280 (King et al., 2004), and phosphorylated Chk1 fails to undergo activation by ATM and the subsequent downstream inactivation of Cdc25 phosphatases that are required for Cdk1 activation (dephosphorylation at Thr14 and Tyr15). Akt also phosphorylates and downregulates the Wee1 family kinases, Wee1 (Katayama et al., 2005) and Myt1 (Okumura et al., 2002), which catalyse the Cdk1-inactive phosphorylations at Thr14 and Tyr15. Akt has been shown to phosphorylate p27 and promotes its degradation (Liang et al., 2002) but it enhances polo-like kinase levels by the inactivation of checkpoint protein, CHFR (Shtivelman, 2003). We found that inhibition of ERK1/2 and Stat3 had no significant effect on Nrf2+/+ and Nrf2−/−GSH cell proliferation (Figure 6). In contrast, blocking Akt signaling with either a PI3K inhibitor or an Akt-specific inhibitor dramatically blocked the Nrf2−/− cell proliferation induced by GSH. In agreement with these results, both the PI3K inhibitor and the Akt-specific inhibitor also blocked wild-type cell proliferation. Taken together, these results strongly suggest that Akt signaling is important in GSH-induced Nrf2−/− cell proliferation. Although GSH supplementation restored the Akt activation, that was deregulated in Nrf2−/− cells, whether GSH directly or indirectly regulate this process remains unclear and warrants further investigation. Akt and ERK1/2 MAP kinases are also implicated in cell-cycle progression at the G1/S transition (Meloche and Pouyssegur, 2007). Depletion of GSH from Nrf2+/+ cells caused inhibition of Akt and ERK1/2 kinase activation and resulted in cell-cycle arrest at the G1/S transition (Figure 7). This arrest was correlated with an impaired colocalization of active MPF complexes (Figure 7). These results suggest that cells may require higher GSH levels for G2/ Oncogene. Author manuscript; available in PMC 2009 February 23.

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M transition as demonstrated by the results obtained in Nrf2−/− cells, in which diminished levels of GSH caused G2/M-phase arrest. However, complete depletion GSH in Nrf2+/+ cells resulted in arrest mainly at the G1 phase, indicating that certain levels of GSH are required for cell-cycle progression at the G1/S transition. These results are in agreement with previous studies using BSO that have pointed to an important role for GSH in cell-cycle progression and proliferation (Conour et al., 2004).


GSH, acting by protein glutathionylation, is being recognized as a potential modulator of the activities of the redox-sensitive thiol proteins, especially those that are involved in signal transduction, and are known to regulate cell growth, differentiation and cell-cycle progression (Fratelli et al., 2004, 2005; Ghezzi et al., 2005; Shelton et al., 2005; Dalle-Donne et al., 2007b; Gallogly and Mieyal, 2007). These proteins include: Ras (Clavreul et al., 2006), ERK1/2 (Ward et al., 2000; Marshall et al., 2002), MEKK1 (Cross and Templeton, 2004), tyrosine phosphatases (Townsend et al., 2006), p53, NF-κB, c-Jun and c-Fos (Klatt and Lamas, 2002; Reynaert et al., 2006; Velu et al., 2007). Thus, it is likely that diminished levels of GSH in Nrf2−/− cells and GSH-depleted Nrf2+/+ cells may cause dysregulation of other signal transduction pathways and the effector transcription factor activation required for gene expression. Consistent with this hypothesis is the fact that mRNA expression profiling has revealed a lack of expression of genes related to cell-cycle progression and proliferation in Nrf2−/− cells, together with the observation that GSH supplementation can restore the expression of several of them (Reddy et al., 2007b). The genes involved in cell proliferation include: integrins, occludins, junction proteins, cadherens and transcription factors as well as growth factors. An Nrf2 deficiency is associated with enhanced expression levels of genes involved in cell-cycle checkpoints' regulation, including the cyclin-dependent kinase inhibitor 2D (Cdkn2d, p19) and promyelocytic leukemia (PML). Cdkn2d inhibits Cdk4 leading to cellcycle arrest at the G1/S transition (Buchold et al., 2007; Laine et al., 2007), whereas PML recruits Chk family kinases and p53 into the PML nuclear bodies, enhances the p53/Chk2 interaction (Louria-Hayon et al., 2003) and also orchestrates a nuclear tumor suppressor network that inactivate nuclear pAkt (Trotman et al., 2006). Cenp-F and Cenp-E have been implicated in spindle checkpoint regulation, kinetochore–microtubule capture and chromosome alignment and stability (Parra et al., 2002; Putkey et al., 2002). We have found that an Nrf2 deficiency enhanced the expression levels of Cenp-F and Cenp-E, but GSH supplementation greatly decreased their expression in Nrf2−/− cells, restoring to levels similar to those seen in wild-type cells (Reddy et al., 2007b). Likewise, GSH supplementation attenuated the elevated levels of septin, a regulator of cytokinesis (Kissel et al., 2005), and Brca1, a regulator of DNA damage repair (Kim et al., 2006), G1/S transition and centrosome duplication (McAllister and Wiseman, 2002), in Nrf2−/− cells. Although these results suggest that Nrf2-regulated GSH-induced redox signaling is important in regulating cell-cycle progression, the mechanisms by which GSH regulates the expression of these and other genes remain to be investigated. Nrf2 associates with Kelch-like ECH-associating protein 1 (Keap1), which is known to sequester Nrf2 into proteosomal degradation under physiologic conditions. Oxidant and toxic insults disrupt the sequestration of Nrf2 by Keap1 thereby triggering the translocation of Nrf2 into nucleus where it regulates the expression of cytoprotective and antioxidant enzymes/ proteins. Keap1 null mice constitutively express cytoprotective and antioxidant enzymes/ proteins (Wakabayashi et al., 2003). Recent reports have demonstrated somatic mutations in Keap1 in cancer tissues and cell lines (Padmanabhan et al., 2006; Singh et al., 2006) and elevated levels of Nrf2 expression. Moreover, a recent study has shown that mutation in Keap1 affected its repressive activity resulting in nuclear accumulation of Nrf2, and induced constitutive expression of cytoprotective and antioxidant enzymes/proteins leading to enhanced cancer cell proliferation (Ohta et al., 2008). Consistent with these results, several recent studies have demonstrated the elevated levels of ROS and antioxidant gene expression Oncogene. Author manuscript; available in PMC 2009 February 23.

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in human cancer cell lines and have indicated that that suppression of antioxidant gene expression can block cancer cell growth (Trachootham et al., 2006; Dolado et al., 2007). These studies suggest that Nrf2 acts as a proto-oncogene and targeting Nrf2 activity by inhibitors may have a therapeutic value. In summary, the studies described here provide genetic evidence for a novel role for the Nrf2 transcription factor in maintaining genomic stability and integrity by mitigating oxidative stress and rescuing cell-cycle arrest. We have further demonstrated that this process is regulated by GSH-mediated redox signaling, which negatively regulates Atm pathways and positively regulates Akt signaling. We have also made the noteworthy observation that merely suppressing ROS levels is insufficient to restore cell-cycle arrest and that GSH-regulated redox signaling is required for this process. Because oxidative stress-induced cell-cycle deregulation has been implicated in the development of a variety of human disorders and that Nrf2 appears to act as proto-oncogene, our findings related to the signaling induced by Nrf2–GSH may have greater implications for understanding the molecular basis underlying various diseases, including the malignancy.

Materials and methods Cell cultures


Nrf2+/+ and Nrf2−/− mice (Itoh et al., 1997) were maintained under the guidelines of the Institutional Animal Care Use Committee of the Johns Hopkins University Bloomberg School of Public Health. Alveolar epithelial cells were prepared from these mice as previously described (Reddy et al., 2007a) and they were cultured in 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid-buffered Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 10 ng/ml keratinocyte growth factor and antibiotics. Other experimental procedures Details related to 8-oxodeoxyguanosine (8-oxodG) detection, TUNEL assays, cell-cycle analysis, real-time RT–PCR analyses, immunoblotting and immunocytochemistry were provided as Supplementary data.

Acknowledgements This work was supported by NIH grants HL66109, ES11863 and SCCOR P50 HL073994 (to SPR), and NIEHS center grant P30 ES 038819. We acknowledge the help provided for FACS analysis by Becton Dickinson Immune Function Laboratory, Johns Hopkins Bloomberg School of Public Health.


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Figure 1.

Disruption of Nrf2 induces G2/M cell-cycle arrest and DNA lesions. (a) Phase contrast images (top) and cell-cycle distributions (bottom) of the Nrf2+/+, Nrf2−/− and Nrf2−/−GSH cells. The cells were harvested on day 5 and single cell suspensions were subjected to fluorescenceactivated cell sorting (FACS) analysis. Data were generated using FlowJo software as described in ‘Materials and methods’. The ‘Y’ axis represents number of cells, whereas the ‘X’ axis represents the florescence intensity. The fluorescence area under each cell cycle is shaded to better visualize the distribution. (b) The graph represents the average percentages of cells in each phase of the cell cycle for Nrf2+/+, Nrf2−/− and Nrf2−/−GSH cells. The data represent the results of four independent experiments. (c) DNA lesions in Nrf2+/+, Nrf2−/− and Oncogene. Author manuscript; available in PMC 2009 February 23.

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Nrf2−/−GSH cells were detected by staining with avidin–FITC. (d) DNA strand breaks were detected by TdT-mediated dUTP nick end labeling (TUNEL) staining.

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Figure 2.

An Nrf2 deficiency induces activation of the ATM and its effectors. Cells were cultured for 5 days, fixed and incubated with phospho-specific antibodies as indicated. Immunofluorescent staining was performed using antibodies recognizing phospho-specific ATM (red, panel a), p53 (green, panel b), Rb (green, c), Chk1 (green, d), Chk2 (green, e) and Cdc25c (green, f) antibodies. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; blue).


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Figure 3.

Colocalization of Cdk1 and cyclin B1 proteins in Nrf2−/− cells. (a) Coimmunostaining analysis of the active forms of Cdk1 and cyclin B1 proteins. Cells were stained with an antibody recognizing phospho-Cdk1 (Thr161; green) and cyclin B1 (red). (b) Coimmunostaining analysis of inactive Cdk1 and cyclin B1 proteins. Cells were stained with antibodies recognizing phospho-Cdk1 (Tyr18; green) and cyclin B1 (red).

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Figure 4.


Antioxidant N-acetylcysteine (NAC) abolishes DNA lesions but fails to restore Nrf2−/− cell proliferation. (a) Phase contrast images and 8-oxodeoxyguanosine staining of Nrf2−/− cells supplemented with NAC (Nrf2−/−NAC) or GSH (Nrf2−/−GSH). NAC supplementation did not improve the proliferation of Nrf2−/− cells even though DNA lesions were prevented. (b) Immunofluorescent staining of Nrf2−/−NAC cells with antibodies recognizing-phospho-specific ATM (green), p53 (green), Chk1 (green) or Chk2 (green) antibodies. No immunofluorescent staining was observed in Nrf2−/−NAC cells. (c) Activation status of active and inactive forms of Cdk1 in Nrf2−/−NAC cells. Top panel: Nrf2−/−NAC cells were stained with anti-Thr161 Cdk1 (active; green) and anti-cyclin B1 (red) antibodies. Lower panel: cells were stained with antiphospho-Cdk1 (Tyr18; green) and anti-cyclin B1 (red) antibodies.


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Figure 5.

Western blot analysis of cell-cycle proteins. Cell lysates from Nrf2+/+, Nrf2−/−, Nrf2−/−GSH and Nrf2−/−NAC cells were prepared and immunoblotted with antibodies specific for Cdk1, Cdc25c, pRb, and cyclin B1. ERK2 and β-actin were used as loading controls.


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Figure 6.

Disruption of Nrf2 results in dysfunctional Akt signaling. (a) Nrf2+/+ and Nrf2−/−GSH cells were treated with LY294702 (10 μM), Akt-II (10 μM), UO126 (10 μM) or AG490 (10 μM) for 48 h. (b) Nrf2+/+, Nrf2−/− and Nrf2−/−GSH cells were stimulated with platelet-derived growth factor (PDGF; 20 ng/ml) and insulin (50 ng/ml) for 30 min, and cell lysates were prepared and immunoblotted with antibodies as indicated. (c) Real-time PCR analyses of Akt1, Akt2 and Akt3 expression in Nrf2+/+, Nrf2−/− and Nrf2−/−GSH cells.


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NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 7.


Effects of glutathione (GSH) depletion on cell-cycle arrest, Akt and ERK1/2 signaling, and MPF complex formation in Nrf2+/+ cells. (a) Cell-cycle distribution of Nrf2+/+ cells treated with buthionine sulfoximine (BSO) was analysed as in Figure 1a. (b) The graph represents the average percentages of cells in each phase of the cell cycle for Nrf2+/+ cells treated with BSO (n = 3). (c) Nrf2+/+ cells were treated with BSO, and cell lysates were prepared and immunoblotted with antibodies as indicated. (d) Coimmunostaining of the active form of the Cdk1 (green) and cyclin B1 (red) proteins (left panel) and inactive forms of the Cdk1 (green) and cyclin B1 proteins (red; right panel).


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Figure 8.

Model depicting possible mechanisms of Nrf2 deficiency leading to cell-cycle arrest. The unfilled arrow indicates the down regulation of glutathione (GSH) levels.
