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Published ahead of print on July 3, 2003, doi:10.1165/rcmb.2003-0087RC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 779-783, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2003-0087RC

Inhibition of c-Jun N-Terminal Kinase Pathway Improves Cell Viability in Response to Oxidant Injury

Yuchi Li, Yuko Arita, Hshi-chi Koo, Jonathan M. Davis and Jeffrey A. Kazzaz

The CardioPulmonary Research Institute, Winthrop-University Hospital, SUNY at Stony Brook School of Medicine, Mineola, New York

Address correspondence to: Yuchi Li, Ph.D., The CardioPulmonary Research Institute, Winthrop-University Hospital, Suite 505, 222 Station Plaza N, Mineola, NY 11501. E-mail: liyuchi{at}hotmail.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Oxidant insults can lead to apoptotic and nonapoptotic cell death. Lung epithelial cells exposed to high levels of oxygen do not die via apoptosis, but through a much slower, morphologically distinct process involving cell and nuclear swelling. In contrast, H2O2 induces a rapid apoptotic cell death. We first assessed the effect of oxidant exposure on activator protein-1 (c-Jun and Fos) and c-Jun N-terminal kinase (JNK) regulation in MLE12 cells. Both oxidants induced c-Jun and Fos expression, albeit with a different pattern of regulation—hyperoxia (95% O2) induced a biphasic response, whereas H2O2 (500 µM) induced a sustained response. We then examined the role of JNK by Western blot, JNK activity assay, and a pull-down assay and observed an identical pattern of regulation. To assess whether JNK functions in a pro-death or pro-survival capacity, we generated stable cell lines that constitutively express a dominant-negative mutation of JNK resulting in significant inhibition of JNK activity. Inhibition of the JNK pathway in this manner prevented hyperoxic and H2O2-induced cell death. These results demonstrate that hyperoxic cell death is pathway-driven and that both modes of death involve the JNK signaling pathway.

Abbreviations: activator protein-1, AP-1 • digoxigenin-11-dUTP, DIG • c-Jun N-terminal kinase, JNK • murine lung epithelial cells, MLE12 cells • nuclear factor-{kappa}B, NF-{kappa}B • sodium dodecyl sulfate, SDS


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pulmonary oxygen toxicity can be a devastating side effect of ventilatory therapy for critically ill patients, causing severe inflammatory lung injury. Exposure to high concentrations of oxygen (hyperoxia) is toxic to cells, and prolonged hyperoxia is uniformly lethal, except in specially selected, mutant cells (1, 2). Although oxidants are generally thought to induce apoptosis, hyperoxia (and low levels of some oxidants) cause nonapoptotic death of lung epithelial cells in culture. This mode of cell death occurs more slowly than apoptosis and is morphologically distinct—the nuclei and cells swell, rather than shrink. It is also biochemically distinguished from apoptosis because DNA ladders do not form and the nuclei are TUNEL-negative (3). The possibility that this process may be pathway-driven and thus not necrosis was suggested by the observation that nuclear transcription factor-{kappa}B (NF-{kappa}B) is activated when cells die via a nonapoptotic pathway when exposed to hyperoxia (4).

Fos and Jun, major components of the activator protein (AP)-1 transcription factor family, have been shown to play an important role in oxidant signaling, oncogenic transformation, immune responses, cellular differentiation, and apoptosis (5–7). Regulation of AP-1 transcriptional activity is achieved by phosphorylation of proteins. c-Jun N-terminal kinase (JNK, also known as stress-activated protein kinase) partially mediates this phosphorylation (8, 9). JNK is one of the leading candidates to transmit and transduce stress signaling into apoptosis signaling in various cell types, including lung epithelial cells (7, 8, 10, 11). Inhibition of the JNK pathway by using dominant-negative JNK or JNK knockout fibroblasts abrogates apoptosis induced by ultraviolet radiation (12). Conversely, expression of activated JNK in untreated cells results in apoptosis (13). Based on previous studies, we propose that hyperoxic cell death is pathway-driven. In the present study, we examine the temporal activation of AP-1 transcriptional factors and JNK signaling pathway in murine lung epithelial cells (MLE12) exposed to hyperoxia and H2O2. We use a dominant-negative JNK construct to demonstrate that activation of the JNK pathway acts as a pro-death signal in both hyperoxic (nonapoptotic) and H2O2 (apoptotic) epithelial cell death.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
MLE12, DNJNK clones, and vector cells were cultured in Hite's media (RPMI 1640 medium, supplemented with 3 mM sodium selenite, 1 mM hydrocortisone, 1 mM ß-estradiol, 5 µg/ml insulin, 10 µg/ml transferrin, 22 mM L-glutamine, and HEPES [10 mM]) supplemented with 2% fetal bovine serum and antibiotics as described (14). Cells were maintained in 5% CO2 at 37°C. Subconfluent (~ 40% confluent for hyperoxia, ~ 70% for H2O2) cultures were used in all experiments. For hyperoxic exposure, cells were cultured in sealed humidified chambers flushed with 95% O2, 5% CO2. Media and gases were refreshed daily. Control cells were cultured in 95% room air, 5% CO2. For hydrogen peroxide studies, cells were treated with 250 or 500 µM H2O2 (Sigma, St. Louis, MO). Cell viability was assessed by Trypan blue exclusion. Apoptosis was assessed by examining nuclear morphology using the DNA dye Hoechst 33258, followed by visualization on a Nikon Optiphot microscope (Melville, NY) equipped with appropriate filters.

Generation and Screening of JNKDN Cell Lines
The full-length DNJNK cDNA was excised with restriction enzymes NcoI and XbaI from the plasmid construct JNK1APF (15) (kindly provided by Dr. Lin, University of Alabama at Birmingham, Birmingham, AL), and subsequently cloned into pOPRSVI plasmid (Stratagene, La Jolla, CA). The resulting plasmid was then transfected into MLE12 cells with Lipofectamine plus reagent (GibcoBRL, Gaithersburg, MD). MLE12 cells transfected with pOPRSVI serve as empty-vector controls. Stable cells were initially selected based on resistance to G418 (200 µg/ml) and then screened for JNK activity by luciferase reporter gene assay and/or in vitro JNK kinase assay.

Western Blots
All procedures were performed as described (4). The primary antibodies used and dilutions for these experiments are as follows: Phospho-specific c-Jun antibody (KM-1; Santa Cruz Biotechnology, Santa Cruz, CA), diluted 1:500; Phospho-specific SAPK/JNK (Thr183/Tyr185) antibody (New England BioLabs, Beverly, MA), diluted 1: 500; secondary antibody was horseradish peroxidase–conjugated goat anti-rabbit IgG (New England BioLabs) diluted 1:1,000. The immunoreactive proteins were visualized using the enhanced chemiluminescence kit (New England BioLabs) according to the supplier's instructions. The filters were exposed to Hyperfilm (Amersham Pharmacia Biotech, Inc., Piscataway, NJ).

JNK Activity Assay
JNK activity was assayed using a SAPK/JNK assay kit from New England BioLabs according to the supplier's instructions. Briefly, proteins were isolated from cell lysate and concentrations determined as described (4). An equal amount of protein was incubated with 2 µg of c-Jun fusion protein beads overnight at 4°C. After several washes with 1x cell lysis buffer and kinase buffer (provided by New England BioLabs), beads were resuspended in 20 µl of kinase buffer supplemented with 100 µM ATP and incubated 30 min at 30°C. The reaction was terminated with 10 µl of 3x sodium dodecyl sulfate (SDS) sample buffer (1x SDS buffer: 62.5 mM Tris, pH6.8, 2% SDS, 10% glycerol, 50 mM DTT). Samples were boiled for 5 min and separated by 10% SDS-PAGE. The Western blotting was performed as described above. The phosphorylated GST–c-Jun fusion protein was detected using anti-phosphorylated c-Jun antibody.

Northern Blot
Total cellular RNA was prepared from untreated controlcells and oxygen- and H2O2-exposed cells using RNA STAT-60 (TEL-TEST"B", Friendswood, TX). Twenty micrograms of RNA from each sample was loaded onto a 2.2-M formaldehyde, 1% agarose gel. After electrophoresis, the RNA was blotted onto a nylon membrane (Millipore, Bedford, MA). Equal loading and RNA integrity was confirmed by visualizing 28S and 18S on gels after electrophoresis and on blots after transfer. For Fos detection, blots were incubated overnight at 50°C with a rat Fos cDNA probe (16) labeled with digoxigenin-11-dUTP (DIG; Boehringer Mannheim, Indianapolis, IN) according to the supplier's instructions. Hybridization and washing were performed as described previously (17). Filters were then incubated with an anti-DIG antibody (1:20,000) (Boehringer Mannheim). Bands were visualized using the enhanced chemiluminescence reagent kit, CDP-star (Boehringer Mannheim), and exposing the filter to Kodak X-ray film. For c-Jun detection, the filter was hybridized with a DIG-labeled rat c-Jun cDNA (16) labeled as above. Both Fos and Jun cDNA clones were kindly provided by Dr. T. Curran (St Jude's Children's Research Hospital, Memphis, TN).

Statistical Analysis
All data are reported as mean ± SD. Statistical analysis was performed using Student's t test or ANOVA with Fisher PLSD post hoc analysis using StatView version 5.01 (SAS Institute, Cary, NC). P values were considered significant at < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Because the regulation of AP-1 activity occurs at both the transcriptional and post-transcriptional levels, we assayed AP-1 mRNA abundance and protein phosphorylation as an indicator of activity. Figure 1 shows a Northern blot of RNA from MLE12 cells exposed to hyperoxia (95% O2) for various times. fos mRNA was not detected in control cells cultured in room air. During continuous exposure to 95% O2 there was a biphasic increase. The first was transient, occurring at 30 min and returning to baseline by 1 h. The second increase generally occurred at 24 h. However, the timing of this event was variable, sometimes being observed as early as 16 h (data not shown). This increased abundance of fos mRNA persisted for several days when cells were maintained in hyperoxic conditions (data not shown). Induced expression of Jun mRNA was also biphasic: a transient increase was seen at 30 min, which returned to near baseline levels after 1 h. The second phase of Jun induction occurred at 16 h, and was more robust than at 30 min. Like fos, the increase in Jun mRNA persisted for several days (data not shown).



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  		Figure 1. Fos and Jun mRNA levels increase in hyperoxia. Subconfluent MLE12 cells were exposed to room air (C) or to 95% O2 for the times indicated. Twenty micrograms of total RNA were electrophoresed on a 2.2-M formaldehyde/1% agarose gel, stained with ethidium bromide to visualize 28S and 18S rRNA, blotted, and hybridized with DIG-labeled Fos (top panel) and c-Jun (bottom panel) cDNA probes. The corresponding 28S and 18S rRNA for these gels are shown below each blot. Results are representative of three independent experiments.

 
Activation of c-Jun requires phosphorylation on both ser-63 and ser-73 with phosphorylation frequently occurring at additional sites as well (15, 18–20). To determine whether c-Jun is phosphorylated during hyperoxia, we performed a Western blot using an antibody specific for c-Jun phosphorylated at ser-63. Figure 2A demonstrates a biphasic pattern of c-Jun phosphorylation that was nearly identical in timing to that observed for c-Jun mRNA. A transient increase occurred within 30 min and a more persistent increase was evident after 16 h of exposure. Three migrating bands were detected, a pattern that was consistently observed in independent experiments.



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  		Figure 2. Oxidants induce Jun phosphorylation and JNK activation. MLE12 cells were cultured in 95% O2 (left) or in 250 µM H2O2 (right) for the times indicated. Protein (20 µg/lane) was electrophoresed, blotted, and incubated with an anti-phosphorylated Jun antibody (A and D) or an anti-phosphorylated JNK antibody (B and E, C and F). JNK activity was confirmed by a JNK immunoaffinity assay performed using 200 µg (C) or 250 µg (F) cell lysates per the manufacturer's instructions (New England Biolabs). Blots are representative of a minimum of three experiments.

 
As a key member of the MAP kinase family, JNK is usually responsible for the phosphorylation of c-Jun in the stress-activated signal transduction pathway (21, 22) leading us to investigate the activity and potential role of JNK during hyperoxia. First, activated JNK was assayed by Western blotting using an antibody directed against the phosphorylated form of JNK (15, 23, 24). Figure 2B shows that expression of phosphorylated forms of JNK p54 and p46 increased after 16–24 h of exposure to hyperoxia. The second approach used an in vitro kinase assay. Figure 2C illustrates that JNK activity was low in control cells and increased dramatically after 16 h of exposure to hyperoxia. This increase in activity persisted for several days (data not shown).

Although hyperoxic death typically is nonapoptotic, these cells do have the capacity to undergo apoptosis when exposed to relatively high levels of oxidants (3, 4, 25). Moreover, p42/p44 MAPK is activated in H2O2-induced apoptosis, but not in nonapoptotic death from hyperoxia (4). Experiments were performed to determine if JNK activation was a distinguishing feature of nonapoptotic death in these cells. Phosphorylated c-Jun increased after 30 min exposure to H2O2, which persisted for ~ 2 h (Figure 2D). Western blot analysis (Figure 2E) and immunoaffinity pull-down (Figure 2F) confirmed that this phosphorylation was due to JNK activation. By 5 h, when apoptosis was already evident (Figure 3C), the level of phosphorylated c-Jun and JNK was reduced to undetectable levels. Thus, activation of the JNK pathway occurs in both nonapoptotic and apoptotic oxidative cell death.



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  		Figure 3. Expression of a dominant-negative JNK protects cells from oxidant injury. (A) JNK activity in MLE12 and DNJNK cell lines DNJ#1 and DNJ#2. A JNK immunoaffinity assay performed on 250 µg cell lysates from MLE12 and DNJNK cells exposed to room air or 95% O2 for 24 h demonstrating a reduction in both baseline and induced JNK activity. (B and C) Viability in hyperoxia and H2O2. DNJNK, empty vector, and MLE12 cells were exposed to 95% O2 for 3 d (B) or 500 µM H2O2 for 4 h (C) and cell viability assessed by dye exclusion of adherent cells. Values are expressed as the percent of viable cells relative to room air control (before treatment) and represent the mean ± SD of triplicate samples. *P < 0.01. (D) Nuclear morphology. Fluorescence micrographs of MLE12 and DNJ#2 were grown on coverslips in room air (control) or exposed to 95% O2 for 3 d or 500 µM H2O2 for 3 h, then fixed and stained with the DNA dye Hoechst 33,258. Note the moderation of nuclear enlargement (hyperoxia) and shrinkage (H2O2) in DNJ#2 nuclei compared with MLE12 nuclei.

 
To further assess the role of JNK pathway in cell death induced by hyperoxia, we generated dominant-negative JNK1/APF-expressing cells (DNJNK). Clones were screened using AP-1 luciferase reporter gene assay and/or in vitro JNK assay (immunoaffinity pull-down). Two clonal isolates, DNJ#1 and DNJ#2, exhibited lower basal and inducible JNK activity compared with MLE12 and/or mixed empty vector clonal cells (Figure 3A). We then exposed these cells to 95% O2 for 3 d and cell viability was assayed by dye exclusion. Exogenous expression of DNJNK significantly improved survival 4- to 5-fold relative to MLE12 (P < 0.01) and 2- to 3-fold relative to Vector cells (P < 0.01) when exposed to O2 (Figure 3B). Next, we examined if DNJNK cells are also resistant to H2O2-induced apoptosis. MLE12 cells, Vector cells, DNJ#1, and DNJ#2 were treated with 500 µM H2O2 for 4 h. Only 20–30% of control cells (MLE12 and Vector) survived this exposure. In sharp contrast, there was no cell death in DNJ#1 cells and ~ 90% survival in DNJ#2 (Figure 3C, P < 0.01). Although the two DNJNK cell lines have different levels of inducible JNK activity, there was no statistical difference in survival between the two cell lines, suggesting that a moderate reduction in JNK activity to a critical value is sufficient to improve cell viability. These data demonstrate that JNK plays a central role in regulating both hyperoxia and H2O2-induced cell death.

Next, we examined if disrupting the JNK pathway would change the mode of cell death. Figure 3D shows that after 3 d in hyperoxia, MLE12 cells and DNJNK cells swell and the nuclei enlarge, similar to the appearance observed in A549 and Hela cells (4, 26). In contrast, both MLE12 and DNJNK cells become smaller in size and their nuclei condense after being exposed to 500 µM H2O2 for 3 h, which is a typical presentation of apoptotic cell death (Figure 3D). Thus, there is no change in the mode of cell death due to the interruption of JNK expression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although there are reports of JNK activation by oxidant stress in various types of cells, limited studies have been done on JNK activation in alveolar epithelial cells (7, 11). In this report, we define and compare the activation of AP-1 transcription factors and JNK in response to hyperoxia and H2O2 in lung epithelial cells. With hyperoxia, induction was biphasic: initially transient and then persistent. The initial, transient induction was not sufficient to induce cell death (data not shown). The persistent phase lasted for several days and involved phosphorylation of c-Jun, an effector of the JNK signaling pathway, which induced cell death. The role of the transient activation remains to be determined. However, it is believed that transient activation is protective (27), whereas prolonged activation leads to sustained c-Jun–mediated transcription of genes that are involved in cell death (9) and/or are required for activating the mitochondrial-dependent death pathway that mediates stress-induced apoptosis (10). Consistent with this model, the activation in response to H2O2 was persistent and induced apoptotic cell death. Several studies have demonstrated induction of AP-1/JNK signaling pathway in response to oxidants and radiation resulting in apoptosis in a variety of cell types (14, 16, 28). The DNJNK cell lines further demonstrate that JNK activation also plays an important role in non-apoptotic programmed cell death. Inactivation of JNK by a dominant-negative mutation abolishes its ability to phosphorylate downstream transcription factors such as c-Jun and ATF-2, thus interrupting normal JNK activation pathway. Both DNJNK cell lines exhibited resistance to hyperoxia-induced nonapoptotic death and to H2O2-induced apoptotic cell death. The observation that apoptosis was not induced despite the AP-1/JNK activation is consistent with the notion that the JNK pathway is involved in both modes of cell death. Taken together, these results demonstrate that these cells are not deficient in AP-1/JNK apoptosis signaling pathway, and suggest that hyperoxia induces factor(s) that inhibit downstream apoptotic events.

DNJNK cells do die during prolonged oxidant exposure. This suggests that the time frame necessary to accumulate pro-death factors is merely extended in these cells. The construct used to derive these cell lines, DNJNK1 (APF), has been reported to prevent activation of JNK (15). However, in the DNJNK cell lines JNK activity was not completely abolished (Figure 3A). Strategies to abolish JNK activity in MLE12 cells are currently under investigation.

Studies from other laboratories have shown that activation of JNK-signaling pathway was sufficient to induce rapid cytochrome c release and apoptosis. Proapoptotic members of the Bax protein subfamily were part of JNK-signaling pathway (13, 29, 30) and in hyperoxia-induced cell death in the mouse lung (31). The status of cytochrome c and the role of Bax/Bcl2 family members in hyperoxic cell death in the DNJNK cell lines remains to be defined.

A recent publication by Zhang and coworkers demonstrated that ERK1/2 plays an important role in hyperoxia-mediated cell death (32). In their studies, hyperoxia induced apoptosis after 72 h with no detectable JNK activity in MLE12 cells. There are several possibilities for the differences, including confluence (quiescent versus growing), and the media used that have been shown to impact the mode of cell death and signaling pathways (33, 34). Furthermore, it is common for multiple pathways to be activated ("crosstalk") in response to a stimulus. The activation, interaction, or redundancy of these pathways (in differing culture conditions) remains to be elucidated.

Several studies have demonstrated that both NF-{kappa}B and JNK play crucial roles in regulating cell survival. Based on studies on tumor necrosis factor-{alpha}–induced JNK activation in fibroblasts, one current hypothesis on how JNK activation contributes to cell death is "breaking the brake on apoptosis," i.e., activated JNK inactivates suppressors of the apoptotic machinery (10, 27). In those studies, activated NF-{kappa}B prevents prolonged JNK activation and cell death. In p65 knockout (RelA-/-) fibroblast cells, tumor necrosis factor-{alpha} induces prolonged JNK activation and subsequent cell death. Similarly, our studies have demonstrated activation of NF-{kappa}B in hyperoxia-exposed A549 cells (4) and that RelA-/- fibroblasts were more sensitive to hyperoxia than their wild-type counterparts (32). Based on these studies, it is likely that hyperoxia induces both survival (NF-{kappa}B) and death pathways (JNK) in lung epithelium, which leads to programmed oncotic cell death manifested by cellular enlargement. The role and/or interaction between NF-{kappa}B and JNK in this cell line remain to be determined.

Current thinking about cell death is dichotomous: cells die either by apoptosis or necrosis. Similarly, a widely held opinion is that apoptosis follows a series of events that are genetically programmed and necrosis does not. Following this reasoning, necrosis is currently the term used to describe slow death associated with swelling from hyperoxia or moderate concentrations of oxidants (3), and very rapid cell death occurring at extremely high oxidant levels (3), as well as almost every other form of unscheduled death consequent to a variety of catastrophic insults. Whether the precise mechanism of the swelling caused by hyperoxia is due to hypertrophy as described in vivo (36) or changes in the electrolyte concentrations, it is conceivable that there are other modes of programmed cell death in addition to apoptosis. Indeed, this and other studies (4, 37) clearly demonstrate that hyperoxic cell death is pathway-driven. Several commentaries have offered different names for death that share only some of the features of apoptosis, e.g., "necrapopotsis" (38). Majno and Joris introduced the term "oncosis" (from the Greek onco for swelling) to delineate between cells that are dying in a morphologically-different manner from apoptosis, because pathologists use "necrosis" to describe dead tissues or cells in a living organism (39). Therefore, using morphology as a guide, we term this form of cell death programmed oncosis.


   Acknowledgments
 
This work was supported in part by grants from the American Lung Association (RG-048-N, Y.L.; RG-062-N, Y.A.), the National Institutes of Health (HL64158, J.M.D) and the Cystic Fibrosis Foundation (J.A.K.). Y.L. is an Edward Livingston Trudeau Scholar. The authors thank Dr. L. L. Mantell and S. Horowitz for their support.

Received in original form March 17, 2003

Received in final form June 5, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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