Deficiency in the c-Jun NH2-terminal kinase signaling pathway confers susceptibility to hyperoxic lung injury in mice

Danielle Morse,1 Leo E. Otterbein,1 Simon Watkins,2 Sean Alber,2 Zhihong Zhou,1 Richard A. Flavell,3 Roger J. Davis,4 and Augustine M. K. Choi1

1Division of Pulmonary, Allergy and Critical Care Medicine, 2Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213; 3Howard Hughes Medical Institute and Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520; and 4Howard Hughes Medical Institute and Program in Molecular Medicine, Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605

Submitted 14 November 2002 ; accepted in final form 17 March 2003


    ABSTRACT
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hyperoxia generates an oxidative stress in the mouse lung, which activates the major stress-inducible kinase pathways, including c-Jun NH2-terminal kinase (JNK). We examined the effect of Jnk1 gene deletion on in vivo responses to hyperoxia in mice. The survival of Jnk1-/- mice was reduced relative to wild-type mice after exposure to continuous hyperoxia. Jnk1-/- mice displayed higher protein concentration in bronchoalveolar lavage (BAL) fluid and increased expression of heme oxygenase-1, a stress-inducible gene, after 65 h of hyperoxia. Contrary to other markers of injury, the leukocyte count in BAL fluid of Jnk1-/- mice was markedly diminished relative to that of wild-type mice. The decrease in BAL leukocyte count was not associated with any decrease in lung myeloperoxidase activity at baseline or after hyperoxia treatment. Pretreatment with inhaled lipopolysaccharide increased BAL neutrophil content and extended hyperoxia survival time to a similar extent in Jnk1-/- and wild-type mice. Associated with increased mortality, Jnk1-/- mice had increased pulmonary epithelial cell apoptosis after exposure to hyperoxia compared with wild-type mice. These results indicate that JNK pathways participate in adaptive responses to hyperoxia in mice.

hyperoxia; mitogen-activated protein kinase; oxidative stress; stress response; oxygen toxicity; apoptosis


EXPOSURE TO HYPEROXIA, or elevated physiological oxygen tension (PO2), causes oxidative lung injury (4). The treatment of advanced lung disease often requires enhanced inspired PO2 to maintain oxygen delivery to peripheral tissues, which paradoxically exacerbates lung damage. An increased production of reactive oxygen species by mitochondria may account for the toxicity of hyperoxia (7, 11). Mice exposed to high PO2 develop a condition similar to human acute respiratory distress syndrome, displaying signs of lung injury by 64–72 h, and generally dying within 90–100 h of continuous exposure (2). Hyperoxia induces endothelial leakage, edema, and inflammatory cell influx into the airways, resulting in diffuse alveolar damage.

Eukaryotic cells respond to stress conditions, such as hyperoxia, with adaptive genetic responses. The mitogen-activated protein kinase (MAPK) signal transduction pathways, which comprise at least three distinct families, represent ubiquitous inducible mechanisms of eukaryotic cell regulation. Among these, the extracellular signal-regulated kinase (ERK) pathway responds to activation by mitogenic stimuli such as hormones and growth factors (i.e., insulin). The c-Jun NH2-terminal kinase (JNK) or stress-activated protein kinase pathways and the p38 MAPK pathways respond preferentially to environmental stress (i.e., oxidants, ultraviolet radiation) and proinflammatory cytokines (5, 9, 15, 16). The JNKs originate from at least three distinct genes (Jnk1, Jnk2, Jnk3), although multiple isoforms may arise from alternative splicing events (8, 10, 15, 16). Although the functions of the p38 MAPK and JNK pathways remain incompletely understood, these pathways are known to regulate cell growth, apoptosis, and inflammatory response (6, 14, 23, 26).

Previously we have shown that hyperoxia induces apoptotic cell death in a murine macrophage cell line (RAW 264.7), which depends on the activation of the ERK signaling pathway (22). Oxidative stress is known to induce all three major MAPK pathways (ERK, JNK, p38 MAPK) in various experimental models (8, 14, 15, 28). We hypothesized that the inactivation of the hyperoxia-inducible JNK pathway would alter the in vivo response to hyperoxia. In the current study, we show that gene-deleted mice with either Jnk1-/- or Jnk2-/- geno-type display increased susceptibility to the lethal effects of hyperoxia. This enhanced susceptibility is associated with increased epithelial cell apoptosis but, paradoxically, decreased airway leukocyte influx.


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Animals. Male C57BL/6 mice (6–8 wk) were purchased from Jackson Laboratory (Bar Harbor, ME) and acclimated for 1 wk with rodent chow and water ad libitum. Male Jnk1-/- and corresponding control mice were generated as previously described (14). All animals were housed in accordance with guidelines from the American Association for Laboratory Animal Care and Research Protocols, and all protocols were approved by the Animal Care and Use Committee at University of Pittsburgh and Yale University.

Hyperoxia exposure in mice. The animals were exposed to >98% O2 at a flow rate of 12 l/min in a 3.7-ft3 glass exposure chamber. The animals were supplied food and water throughout the exposure. For survival studies, animals were monitored hourly, and the time of death was noted. For the measurement of markers of lung injury, separate groups of animals were removed from the chamber at predetermined time points and killed under anesthesia.

Lipopolysaccharide exposure in mice. Mice were placed in a wire mesh cage, which was in turn placed inside an airtight Plexiglas chamber. Lipopolysaccharide (LPS) was nebulized into the chamber continuously for 1 h with a model 40 nebulizer (DeVilbiss, Somerset, PA), which generates 5-µm-diameter particles. The aerosol was directed against a deflector plate at the entrance of the chamber to ensure even dispersal of the LPS. The aerosol was drawn through the chamber at a steady rate by a blower motor (Grainger, Baltimore, MD) connected to the exit port.

Measurement of lung injury markers. The Jnk1-/- mice and their controls were removed from the exposure chamber and killed after 65 h of continuous exposure to hyperoxia. The animals were killed with an overdose of ketamine and transection of the heart. Bronchoalveolar lavage (BAL, 35 ml/kg) was performed four times with PBS (pH 7.4). Cell pellets were pooled from the lavages and resuspended in PBS. The supernatant from the first lavage was saved and frozen for protein determination. Cell counts were performed with a Neubauer hemocytometer (VWR, Boston, MA). For differential analysis, samples were cytocentrifuged and stained with Diff-Quik (Fisher Scientific, Pittsburgh, PA). The protein concentration in each sample was determined by a standard Bradford assay (Sigma, St. Louis, MO) using BSA as the standard. Separate animals were used for myeloperoxidase (MPO) determination (13). After exsanguination, the entire left lung was excised and frozen at -70°C for MPO measurement, whereas the right lung was weighed and desiccated in a 60°C oven to determine the wet-to-dry lung weight. The frozen lung samples were homogenized in phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide, sonicated for 15 s, and further disrupted by a freeze-thaw cycle in liquid nitrogen. The extract was centrifuged at 20,000 g for 20 min at 4°C. We assayed the supernatants for MPO activity by measuring the absorbance at 460 nm, using a 0.167 mg/dl solution of o-dianisidine dihydrochloride in 0.0005% hydrogen peroxide as the substrate. MPO activity is expressed per gram of dry weight.

Western analysis of lung JNK phosphorylation. Assay kits were purchased from New England Biolabs (Beverly, MA) and used per manufacturer's instructions. The protocol is described briefly: following exposure to hyperoxia (65 h), lungs from C57BL/6 mice were excised and homogenized in buffer A [25 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leukopeptin, and 1 µg/ml aprotinin]. Protein concentrations were determined with the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Twenty micrograms of protein were boiled for 5 min and loaded into a 12% polyacrylamide gel. Proteins were separated by SDS-PAGE. After SDS-PAGE, the proteins were transferred to a nitrocellulose membrane. Membranes were incubated with blocking buffer [5% nonfat dry milk in 10% Tween in Tris-buffered saline (TTBS)] for 3 h, washed with TTBS, and then incubated overnight in the corresponding rabbit polyclonal primary antibody directed against phosphorylated JNK. The following day, membranes were washed in TTBS, and proteins were visualized with horseradish peroxidase-conjugated anti-rabbit IgG and the enhanced chemiluminescence assay (Amersham Life Sciences, Arlington Heights, IL). All membranes were subsequently stripped using standard stripping solution (100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris · HCl, pH 6.8) at 50°C. Membranes were reprobed with rabbit polyclonal antibody targeting total nonphosphorylated JNK to assure equal loading.

Northern analysis of lung heme oxygenase-1 mRNA. After exposure to hyperoxia (65 h), lungs from Jnk1-/- and wild-type mice were excised and homogenized. Total RNA was extracted from lung homogenates by solubilization in TRIzol lysis buffer (GIBCO BRL, Carlsbad, CA), followed by chloroform extraction. Total RNA (10 µg) was electrophoresed on a 1% agarose gel and then transferred to GeneScreen Plus membrane (DuPont NEN, Boston, MA). The content and integrity of the RNA were confirmed by ethidium bromide staining of the gel. The membranes were prehybridized and then hybridized at 65°C in Church and Gilbert's buffer containing denatured salmon sperm DNA and a 32P-labeled fragment of rat heme oxygenase (HO)-1 cDNA. The cDNA was labeled with the random primer kit (Amersham, Piscataway, NJ). Nylon membranes were washed 2x 25 min at 65°C in wash buffer A (0.5% BSA, 5% SDS, and 1 mM EDTA in 40 mM phosphate buffer, pH 7.0) and then washed 3x 25 min in wash buffer B (1% SDS, 1.0 mM EDTA, and 40 mM phosphate buffer, pH 7.0), followed by autoradiography.

Microscopic analysis of cell death. After 65-h exposure to hyperoxia, lungs were harvested and inflation fixed with 2% paraformaldehyde in PBS and immersed in the same fixative for 1 h. The lungs were then placed in 30% sucrose in PBS overnight and flash frozen in liquid nitrogen-cooled isopentane. Five-micrometer lung cryosections were cut and washed sequentially in PBS, then in PBS containing 0.5% BSA and 0.15% glycine. The In Situ Cell Death Detection Kit (Roche Diagnostics, Mannheim, Germany) was used to detect DNA strand breaks generated during apoptosis. The sections were washed and incubated in a 1:200 dilution of rat anti-mouse macrophage/monocyte antibody (Serotec, Raleigh, NC) for 1 h. After three washes in BSA, the sections were stained with a 1:3,000 dilution of cy3-conjugated AffinPure Goat Anti-Rat IgG (heavy + light chain) (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h, washed, and stained with Hoechst dye (Sigma) for 30 s and mounted in Gelvatol (Monsanto, St Louis, MO). Images were collected with an Olympus Provis microscope (Olympus, Tokyo, Japan) as well as an Olympus Floview scanning confocal microscope.

cDNA array of mouse lung RNA after exposure to hyperoxia. RNA was extracted from the lungs of four Jnk1-/- and four wild-type mice after 65 h of exposure to hyperoxia as described above. The RNA from each group was then pooled. Probe mixtures were synthesized by reverse transcribing both RNA populations with the cDNA primer mix provided by Clontech Laboratories/BD Biosciences (Palo Alto, CA) with the commercially available Atlas cDNA mouse array. The radioactively labeled probe mixes were each hybridized to separate nylon cDNA expression arrays and washed according to the instructions provided. After washing, the hybridization patterns were analyzed by autoradiography. Housekeeping genes included as positive controls on the microarray membrane were used to normalize the hybridization signals. Quantitation of gene expression was performed by Clontech Laboratories/BD Biosciences with proprietary software.

Statistical analysis. Data are expressed as means ± SE. Differences in measured variables between the experimental and control groups were assessed with Student's t-test. Statistical difference was accepted at P < 0.05.


    RESULTS
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 METHODS
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 DISCUSSION
 REFERENCES
 
Effect of hyperoxia on JNK induction in mouse lungs. Jnk1-/- and C57BL/6 control mice were exposed to >98% oxygen continuously, and lungs were harvested at daily time points for analysis of JNK activation. Lungs were homogenized and analyzed by Western blotting for JNK phosphorylation. The level of phospho-JNK was elevated at 48- and 72-h time points relative to nonexposed control mice (Fig. 1). As expected, the level of JNK activation was higher in control mice than in the Jnk1-/- mice. Because the Jnk1-/- mice have an intact Jnk2 gene (and each gene is capable of making the various isoforms of JNK via alternate mRNA splicing), the level of JNK protein is not zero in the gene-deleted mice.



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Fig. 1. Lung JNK activation by exposure to hyperoxia in vivo. Jnk1-/- and C57Bl/6 control mice were exposed to >98% O2 continuously and lungs were harvested at daily time points for Western blot analysis of JNK phosphorylation. The level of phosphorylated JNK (P-JNK) was elevated at 48- and 72-h time points relative to nonexposed control mice. As expected, the level of JNK activation was higher in control mice than in the Jnk1-/- mice. C, control C57Bl/6 mice.

 

Effect of Jnk1 gene deletion on hyperoxia survival time in mice. To determine the effect of Jnk1 on the survival of mice exposed to hyperoxia, we placed mice in an atmosphere of >98% oxygen, and the time of death was recorded. After 80 h of continuous exposure, 86% of wild-type mice (n = 14) remained alive, whereas only 32% of Jnk1-/- were still living (n = 22, Fig. 2, P < 0.05). Jnk2-/- mice demonstrated a similar but less-pronounced susceptibility to hyperoxia (data not shown). The range of survival for Jnk1-/- mice was 68–86 h, for Jnk2-/- 67–94 h, and for wild-type mice 72–125 h.



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Fig. 2. Hyperoxia-induced mortality in Jnk1-/- and control mice. Jnk1-/- and corresponding wild-type (WT) mice were exposed to >98% O2 continuously in exposure chambers and monitored for survival. The survival of the Jnk gene-deleted mice in hyperoxia was markedly reduced relative to wild-type mice. After 80 h of exposure, 86% of WT mice (n = 14) remained alive, whereas only 32% of Jnk1-/- (n = 22) mice remained alive. *P < 0.05 relative to WT control.

 

Effect of Jnk1 gene deletion on hyperoxia-induced lung injury markers. After 65 h of continuous hyperoxia, Jnk1-/- mice displayed a higher concentration of BAL protein, a marker of lung injury (2), relative to wild-type controls (Fig. 3A) (P < 0.05). The 65-h time point was chosen because this was several hours before the Jnk1-/- mice would begin to succumb to hyperoxia. Again, a similar but less-pronounced trend was seen in Jnk2-/- mice (data not shown). The expression of HO-1, an oxidative stress-responsive gene, was also assayed in lung tissue. After 65 h of continuous exposure to hyperoxia (>98% oxygen), total RNA was extracted from whole lungs and analyzed by Northern blotting. The levels of HO-1 mRNA were substantially higher in three lungs taken from Jnk1-/- mice than in three wild-type controls (Fig. 3B), confirming a higher level of oxidative stress in the JNK1 gene-deleted mice.



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Fig. 3. Lung injury markers in Jnk1-/- mice and WT mice subjected to hyperoxia. Jnk1-/- and corresponding control WT mice were exposed continuously to >98% O2. After 65 h of exposure, the lungs were either lavaged for total protein determination in the bronchoalveolar lavage (BAL), or lungs were harvested for RNA extraction. A: the protein content of BAL fluid after exposure to hyperoxia was higher in Jnk1-/- than in WT mice. Data represent the means ± SE of 5 mice in each group. *P < 0.05 relative to WT control. B: expression of heme oxygenase (HO)-1, a stress-induced gene, was higher in the lungs of Jnk1-/- mice compared with WT mice after exposure to hyperoxia. The Northern blot represents RNA from the lungs of 3 independent WT mice (lanes 1–3) and 3 independent Jnk1-/- mice (lanes 4–6).

 

Effect of Jnk1 gene deletion on hyperoxia-induced airway leukocyte content. Jnk1-/- mice, which displayed increased susceptibility to hyperoxia and exhibited higher BAL protein content, had markedly lower levels of leukocytes in BAL than wild-type mice (Fig. 4). The BAL leukocyte counts of Jnk2-/- mice were also significantly reduced relative to wild-type controls, though to a lesser extent than in Jnk1-/- mice (data not shown). The differences in BAL leukocyte counts between the Jnk1-/- or Jnk2-/- mice and control mice were highly statistically significant (P < 0.00001).



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Fig. 4. BAL leukocyte counts in Jnk1-/- and control mice exposed to hyperoxia. Jnk1-/- and corresponding WT mice were continuously exposed to >98% O2. At 65 h, the lungs were lavaged, and leukocyte counts were determined. The leukocyte counts in BAL fluid were decreased in Jnk1-/- mice relative to control mice. The cell count is expressed as cell number (x 104) per ml. Data represent the means ± SE of 13–21 mice in each group. *P < 0.00001 relative to WT control.

 

Lung MPO activity. To determine the relationship between BAL leukocyte count and neutrophil content, we measured MPO activity in the lung before and after exposure to hyperoxic conditions. The Jnk1-/- mice had higher basal MPO activity in the lung than wild-type mice (Fig. 5, P < 0.05). Despite lower leukocyte counts in the BAL, Jnk1-/- mice displayed a similar level of MPO activity to control mice after continuous hyperoxia treatment.



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Fig. 5. Myeloperoxidase (MPO) activity in mouse strains subjected to hyperoxia. Total lung MPO activity was determined in Jnk1-/- and WT mice before and after continuous exposure to >98% O2 for 65 h. Jnk1-/- mice displayed higher pulmonary MPO activity at baseline than did control mice. After exposure to hyperoxia, there was no difference in MPO activity between Jnk1-/- mice and control. Data represent 5 animals in each group. *P < 0.05 relative to WT control; #P < 0.001 relative to untreated mice.

 

Effect of LPS pretreatment on airway neutrophil influx and hyperoxia-induced mortality in mouse strains. To examine whether the difference in BAL leukocyte count between groups of animals is associated with the abnormal leukocyte trafficking and to test whether the increased susceptibility of Jnk1-/- mice is specific to the hyperoxia model, we used nebulized LPS to stimulate neutrophil migration into the airway. BAL neutrophil content was similar in Jnk1-/- and wild-type mice after LPS treatment (Fig. 6A). Previously, it has been shown that LPS pretreatment confers cross-tolerance against subsequent hyperoxia challenge (3, 28). Pretreatment with LPS prolonged the survival of both wild-type and Jnk1-/- mice to a similar extent during continuous hyperoxia exposure (Fig. 6B).



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Fig. 6. Effect of inhalation LPS on airway neutrophil influx (A) and hyperoxia-induced mortality (B) in mouse strains. Jnk1-/- and corresponding WT mice were exposed to inhalation LPS (75 mg nebulized in a volume of 25 ml over 50 min). After LPS treatment, mice were assayed for neutrophil content in the BAL (A) or subjected to continuous hyperoxia (>98% O2) and monitored for survival (B). Inhaled LPS caused an increase in airway neutrophil count in both WT and Jnk1-/- mice (A). Neutrophils were not detected in BAL fluid from untreated mice. The difference in BAL neutrophil numbers between WT and Jnk1-/- mice was not statistically significant. The neutrophil count is expressed as the cell number x 103 per ml. Data represent the means ± SE of 8 mice in each group. *P < 0.05 relative to WT control. B: inhaled LPS protected both WT mice and Jnk1-/- mice against hyperoxia. {blacktriangleup}, WT mice subjected to hyperoxia; {blacktriangleup} Jnk1-/- subjected to hyperoxia; {blacksquare}, WT mice subjected to hyperoxia with LPS pretreatment; {square}, Jnk1-/- subjected to hyperoxia with LPS pretreatment. Data represent 5 animals in the LPS-treated groups and 18–23 animals in the remaining groups.

 

Lung apoptosis in mouse strains exposed to hyperoxia. The JNKs participate in the regulation of stress-induced apoptosis (31). Because hyperoxic stress induces apoptosis in the lungs of mice and rats (1820), we examined the effect of hyperoxia on lung apoptosis in Jnk1-/- mice relative to wild-type mice. Lung sections were obtained from mice exposed to either normoxia or hyperoxia and analyzed by an in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay, which labels the 3'-carboxy termini of DNA fragments generated by apoptosis-associated endonuclease activity (29). After hyperoxic exposure, the total number of TUNEL-positive cells was higher in the lungs from Jnk1-/- mice than those of wild-type mice (Fig. 7, A and B). High-powered microscopic analysis of hyperoxia-injured lungs with specific staining for macrophages was undertaken to determine the predominant cell type undergoing apoptosis. Fluorescent images of TUNEL are overlaid with differential-interference-contrast images to show the structure of the underlying lung tissue. From these images (Fig. 7, C and D), it is apparent that most of the TUNEL-positive cells are epithelial cells.



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Fig. 7. Microscopic analysis of cell death in lung sections after exposure to hyperoxia. Jnk1-/- and corresponding WT mice were exposed continuously to >98% O2. After 65 h of exposure, lungs were harvested and analyzed for apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining. Images were collected with an Olympus Provis microscope as well as an Olympus Floview scanning confocal microscope. A and B: low-power views of control and Jnk1-/- lungs, respectively; the green signal represents TUNEL-positive cells. Jnk1-/-lungs have increased TUNEL staining when compared with controls. C and D: high-power views of control and Jnk1-/- lungs, respectively, with a differential-interferencecontrast overlay to show the structure of the underlying lung tissue. The green signal again represents TUNEL staining, whereas the red signal represents a macrophage-specific stain. Yellow arrows point to macrophages, which are not apoptotic, and white arrows point to epithelial cells, which are TUNEL positive. Most of the TUNEL-positive cells are epithelial. Scale bar in A is 100 µm and 25 µm in C.

 

cDNA array of mouse lung RNA after exposure to hyperoxia. To uncover some of the genes that might contribute to the susceptibility of the Jnk1-/- mice or that might be markers of severity of hyperoxic lung injury, we performed a cDNA microarray. After 65 h of continuous exposure to hyperoxia (>98% oxygen), total RNA was extracted from whole lungs. Microarrays were then probed with cDNA from pooled groups of four Jnk1-/- and four control mice. A total of 79 differentially expressed genes were uncovered; an extract of those data is shown in Fig. 8. Autoradiographs of the hybridized membranes are shown in Fig. 8, bottom.



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Fig. 8. Results of Atlas cDNA array. The table at top lists selected differentially regulated genes from Jnk1-/- and WT mouse lungs after exposure to hyperoxia. Data were generated from the Atlas cDNA microarray. Positive values indicate that gene expression was higher in Jnk1-/- lungs; negative values indicated that gene expression was higher in control lungs. Data represent pooled samples of RNA extracted from the lungs of 4 mice in each group. Bottom: the results of autoradiography of the Atlas cDNA array. Left: hybridization with a probe derived from pooled RNA extracted from the lungs of 4 control mice after exposure to hyperoxia. Right: the corresponding cDNA hybridization from the lungs of 4 Jnk1-/- mice.

 


    DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The JNK pathway responds to activation by proinflammatory mediators and environmental stress conditions (14), including hyperoxia, as we have confirmed here. Little is known, however, about the role of the JNK pathway in lung responses to hyperoxia. The major objective of this study was to examine the effect of Jnk1 or Jnk2 deletions on the hyperoxic response in vivo. Genetically altered mice provide a useful tool for studying the involvement of a gene product in physiological processes. Study of Jnk gene deletion is complicated, however, by redundancy of Jnk1 and Jnk2. Multiple JNK isoforms are produced through alternate splicing of the primary transcripts, and either the Jnk1 or the Jnk2 gene can encode particular isoforms that preferentially target a specific substrate (8). It is possible that in the absence of either Jnk1 or Jnk2, physiological compensation occurs through the activation of parallel pathways. As shown in our Fig. 1, the level of JNK protein in the Jnk1-/- mice is not zero, although the degree of activation by hyperoxia is significantly lower than in control mice. Unfortunately, simultaneous deletion of both Jnk1 and Jnk2 genes cannot be examined in vivo, due to the lethality of this condition in mice. Of note, previous studies in vitro have shown that Jnk1 gene disruption causes a larger decrease in JNK activity than Jnk2 gene disruption (30).

That said, the increased susceptibility of Jnk1-/- or Jnk2-/- mice to hyperoxia suggests that the JNK pathways participate in protective responses to oxidative injury. Hyperoxia-induced lung injury occurs more rapidly and severely in mice lacking the Jnk1 or Jnk2 genes as shown by decreased survival time, increased BAL protein content, and activation of the stress-response gene Ho-1, a general marker of oxidative stress in the lung (1). Deletion of Jnk1, however, does not affect the development of LPS-induced chemotolerance, suggesting that the JNK pathway does not contribute to this process. The mechanisms underlying LPS-induced chemotolerance remain unclear but may involve the upregulation of stress-activated genes such as manganese superoxide dismutase (28).

Although both Jnk1-/- and Jnk2-/- mice proved to be more susceptible to hyperoxia than wild-type mice, this susceptibility was more pronounced in Jnk1-/- mice. This finding is in keeping with the observation by Tournier et al. (30) that Jnk1 gene disruption causes a larger decrease in JNK activity in vitro than that caused by disruption of the Jnk2 gene. Given the more exaggerated response of the Jnk1-/- mice, this strain was used exclusively for many of the above studies.

The discordance between airway leukocyte counts and survival in our model is intriguing. Hyperoxic lung injury is characterized by leukocyte accumulation within pulmonary microvessels and subsequent leukocyte transmigration into the alveolar wall and air spaces (19). A substantial proportion of the leukocytes generally found in the microcirculation and alveolar septae of hyperoxia-exposed lungs are neutrophils (19). The dramatically decreased number of leukocytes in the airways of the Jnk gene-deleted mice in our study was contrary to what would be expected in the setting of increased injury. The JNK pathway has been shown to play a role in TNF-{alpha}-induced expression of E-selectin on endothelial cells (25), which is critical for leukocyte adhesion and infiltration. The results of the MPO assay indicate that the total neutrophil content of the Jnk1-/- mouse lungs is no different from control, supporting the hypothesis that there is a defect in neutrophil margination or trafficking. With this hypothesis in mind, we tested the response to inhaled nebulized LPS, a known inducer of airway neutrophilia. The normal response of Jnk1-/- mice to nebulized LPS suggests that if there is a defect in neutrophil trafficking in these mice, it must be specific to particular stimuli or modes of lung injury.

The JNK pathway is thought to play a major role in cellular apoptosis, but the functional significance of apoptosis in response to hyperoxia remains poorly defined. Mantell et al. (18) reported that the amount of hyperoxia-induced apoptosis in a mouse model correlates with the extent of lung injury. The authors found that a strain of mice resistant to hyperoxia displays a lower level of apoptosis than a hyperoxia-sensitive strain. In contrast, Otterbein et al. (20) reported that older rats display increased tolerance to hyperoxia yet exhibit higher lung apoptotic indexes than rats that were sensitive to hyperoxia. In these studies, the apoptotic index was inversely proportional to the extent of lung injury and correlated with increased survival, opposite to the findings reported here. A major limitation in interpreting such conflicting data arises from the difficulty to determine which specific cell types are responsible for the apoptotic signals in TUNEL assays of lung tissue sections. Our data, which demonstrate that epithelial cells are the predominant cells affected in the hyperoxia-sensitive Jnk1-/- mice, support the notion that increased pneumocyte apoptosis is a consequence of more severe oxidative injury. The baseline elevation of lung MPO activity in Jnk1-/- mice suggests the possibility of impaired neutrophil apoptosis in these mice, but the TUNEL signal from lung leukocytes was low in both Jnk1-/- and control animals.

The broad spectrum of cellular functions that involve JNK signaling gives rise to many potential future avenues of investigation in this model. One method for defining future studies is to begin with an assessment of differentially expressed genes. Gene array technology allows for comparative analysis of genome-wide patterns of mRNA expression from which inferences about overall cell function and the function of individual gene components can be drawn. Using this technique, we have compared the lungs of Jnk1-/- mice and control mice after exposure to hyperoxia and identified 79 differentially expressed genes. The particular commercial array that was used can identify a maximum of 1,176 mouse genes present at an abundance of 10–20 copies per cell or greater. The genes that were identified belonged to several broad functional classes including cell signaling, transcription, growth factors, stress response, and matrix remodeling. Some of the genes that were differentially induced relate to already identified functions of JNK. For instance, T cell deathassociated protein (TDAG51) was downregulated in the Jnk1-/- mouse lungs relative to control. This gene has been shown to play an essential role in induction of apoptosis by coupling T cell receptor stimulation to Fas expression (21), and JNK1 is known to regulate T cell apoptosis and proliferation (27). Another gene, growth arrest and DNA damage-inducible protein 45 was highly upregulated in the Jnk1-/- mouse lungs; this gene has been shown to mediate the activation of the JNK pathway and cytokine production in effector TH1 cells (17). Perhaps more interesting from the point of view of hyperoxic lung injury is the increase in genes involved in tissue remodeling. Several genes related to transforming growth factor-{beta} were differentially expressed in the Jnk1-/- mice, as were insulin-like growth factor binding protein 3, tissue inhibitor of metalloproteinases 2, and several matrix components. Other genes such as {alpha}6-integrin could be more directly involved in the increased susceptibility of the Jnk1-/- mice. The abundance of mRNA for this integrin was fivefold lower in the Jnk1-/- animals than in wild type; this gene has been implicated in apoptosis, cell adhesion to the extracellular matrix, and transmigration of neutrophils across the endothelial cell barrier (12, 24, 25). The differentially expressed genes highlighted by the array do not allow for any specific conclusions to be drawn. Gene array analysis provides a starting point, however, for the development of hypotheses to explain why mice deficient in the JNK MAPK signaling pathway are so susceptible to hyperoxic lung injury.


    ACKNOWLEDGMENTS
 
The work by A. M. K. Choi was supported in part by National Institutes of Health Grants HL-60234, HL-55330, and AI-42365. L. Otterbein was supported by an American Heart Association grantin-aid.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. M. K. Choi, Div. of Pulmonary, Allergy and Critical Care Medicine, NW 628 UPMC Montefiore, 3459 5th Ave., Pittsburgh, PA 15213 (E-mail: choiam{at}msx.upmc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
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 ABSTRACT
 METHODS
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 DISCUSSION
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