Reactive oxygen species (ROS) can cause cell injury and death via mitochondrial-dependent pathways, and supplementation with antioxidants has been shown to ameliorate these processes. The c-Jun NH2-terminal kinase (JNK) pathway has been shown to play a critical role in ROS-induced cell death. To determine if targeting catalase (CAT) to the mitochondria provides better protection than cytosolic expression against H2O2-induced injury, the following two approaches were taken: 1) adenoviral-mediated transduction was performed using cytosolic (CCAT) or mitochondrial (MCAT) CAT cDNAs and 2) stable cell lines were generated overexpressing CAT in mitochondria (n = 3). Cells were exposed to 250 μM H2O2, and cell survival, mitochondrial function, cytochrome c release, and JNK activity were analyzed. Although all viral transduced cells had a transient twofold increase in CAT activity, MCAT cells had significantly higher survival rates, the best mitochondrial function, and lowest JNK activity compared with CCAT and LacZ controls. The improved protection with MCAT was observed in primary type II lung epithelial cells and in transformed lung epithelial cells. In the three stable cell lines, cell survival directly correlated with extent of mitochondrial localization (r = 0.60572, P < 0.05) and not overall CAT activity (r = −0.45501, P < 0.05). Data indicate that targeting of antioxidants directly to the mitochondria is more effective in protecting lung epithelial cells against ROS-induced injury. This has important implications in antioxidant supplementation trials to prevent ROS-induced lung injury in critically ill patients.
- reactive oxygen species
- c-Jun NH2-terminal kinase
- c-Jun NH2-terminal kinase pathway
primary lung disease, the associated inflammatory response, and treatment with supplemental oxygen all result in the generation of increased amounts of reactive oxygen species (ROS), which can cause significant damage to the lung. The susceptibility of the lung to oxidative injury depends largely on the ability to upregulate protective ROS scavenging systems. Unfortunately, both intracellular and extracellular antioxidants are expressed at relatively low levels in the human lung and are often not acutely induced when exposed to oxidative stress. Increasing evidence suggests that oxidative injury is intimately involved in the pathogenesis of acute and chronic lung injury in infants, children, and adults (7). Animal models and clinical studies of adults with acute respiratory distress syndrome (ARDS) reveal biochemical evidence of significant ROS-induced lung injury (11). Thus development of therapeutic strategies to supplement endogenous antioxidant enzyme (AOE) activities to scavenge excess ROS represents a rational approach to minimizing lung injury caused by oxidant stress.
AOE such as superoxide dismutase (SOD; converting superoxide anion into H2O2) and catalase (CAT; converting H2O2 into water) are appropriate agents for augmentation of antioxidant defenses in the lung. We have previously shown that pulmonary epithelial cells overexpressing SOD have significantly improved survival when exposed to oxidative stress such as hyperoxia and paraquat (12, 14). Numerous in vivo and in vitro studies have indicated that AOE can consistently protect cells from injury and death in response to oxidant stress, although specific strategies to optimize this protection have not been definitely established.
The apoptotic process is regulated by many intracellular signaling pathways, including the c-Jun NH2-terminal kinase (JNK) pathway (5, 8, 18, 26). Both in vitro and in vivo studies have demonstrated that JNK can function as a proapoptotic kinase (8, 18). jnk1−/−jnk2−/− mice have been shown to be resistant to apoptosis induced by ultraviolet (UV) irradiation and anisomycin, with embryonic fibroblasts from these mice also resistant to UV irradiation-induced apoptosis (29). The role of mitochondria as stress sensors and executioners has been studied intensively. Damage to the mitochondrial membrane by ROS leads to a loss of membrane potential and pore opening, causing swelling, leakage of cytochrome c (cyt c), and initiation of apoptosis (22, 30, 33). The absence of JNK causes a defect in the mitochondrial death signaling pathway, including the failure to release cyt c (8). We have also demonstrated previously that downregulation of the JNK pathway protected murine lung epithelial cells with alveolar type II cell characteristics (MLE12) from H2O2-induced apoptosis (17). This suggests that JNK is an intrinsic component of the mitochondrial-dependent death pathway during stress-induced apoptosis.
Recently, there has been intense interest in determining whether specifically protecting the mitochondria can optimally improve cell survival in response to oxidant injury (compared with cytosolic localization; see Ref. 23). For the current study, we hypothesized that overexpression of AOE specifically targeted to mitochondria should optimally protect this important organelle and in turn protect lung epithelial cells from oxidant injury. This concept was tested in transformed murine lung epithelial cells (MLE12) and in primary type II lung epithelial cells. Cell survival, markers of mitochondrial integrity and function, and activation of the JNK pathway were analyzed in response to H2O2 in cells overexpressing CAT in either the cytosol or mitochondria, using stable or transient transduction techniques.
MATERIALS AND METHODS
MLE12 cells (ATCC, Manassas, VA) were cultured in a modified Hite's media supplemented with 2% FBS and antibiotics as previously described (31). Cells were maintained in 5% CO2 at 37°C. Subconfluent (∼50–70%) cultures were used in all experiments. For H2O2 studies, cells were treated with either 250 μM H2O2 (for transduced MLE12 cells; Sigma, St. Louis, MO) or 1 mM H2O2 (for primary cells). Cell viability was assessed by trypan blue dye exclusion, which correlates well with several other cell viability techniques (12).
Isolation of primary alveolar type II cells.
Type II cells were isolated from lungs of specific pathogen-free male Sprague-Dawley rats (200–300 g) according to a previously published protocol (4, 10). Briefly, rats were anesthetized (50 mg/kg Nembutal) and ventilated after tracheostomy. The lungs were cleared of blood by perfusion of buffered saline through the pulmonary artery and harvested en bloc with trachea and airways. The harvested lungs were treated (3 times) for 10–12 min with intratracheally instilled elastase solution (3 U/ml). Thereafter, the elastase activity was inhibited with fetal bovine serum, and the lungs were chopped into 1-mm3 pieces on a McIlwain tissue chopper. The lung mince was shaken vigorously in the presence of DNase I to obtain free cells. The cells were sequentially filtered through nylon mesh filters (170, 37, and 15 μm) to remove cell aggregates. The free cells in the filtrate were sedimented, counted, and cultured for 1 h on IgG-coated bacteriological plates to remove macrophages and other Fc receptor-containing cells. Type II cells in suspension were harvested by “panning” and centrifuged for 10 min at 300 g. The cell pellet was suspended in minimal essential medium (MEM) containing 10% fetal bovine serum. Cell counts and viability analyses showed that these were routinely 85–90% type II cells as determined by phosphine staining of lamellar bodies. Greater than 95% of these cells excluded trypan blue. Cells were then cultured for indicated periods in MEM containing 10% FBS.
Recombinant adenoviral-mediated expression of CAT.
Recombinant adenovirus (rAd) was used for the transient induction of CAT expression (transduction). Viral transduction was performed with type 5, replication-deficient adenovirus. Adenoviral constructs were generated and grown at the University of Iowa Vector Core Laboratory, carrying either a human CAT cDNA (rAd.CMVCAT) or a chimeric cDNA composed of the human MnSOD mitochondrial localization signal and human CAT cDNA (rAd.CMVMCAT). An rAd harboring the bacterial enzyme β-galactosidase (rAd.lacZ) was used for transduction controls. These constructs contain the cytomegalovirus (CMV) enhancer/promoter and SV40 polyadenylation site for efficient transgene expression. MLE12 cells were seeded at 1.5–1.7 × 105/10 cm dish and then transduced with the various rAd particles at a multiplicity of infection (MOI) ranging from 100 to 200 viral particles/cell in complete media. Cells were washed, and fresh media was added after 20 h. Cells were then seeded at 3.0–4.0 × 105/well in six-well plates, allowed to adhere overnight, and then exposed to H2O2. Enzymatic activity was assayed at the time of experiment in duplicate cultures. Primary cells were seeded at 5 × 105/well on a 24-well plate. Postseeding (48 h), cells were transduced with the various rAd particles at an MOI of 6 plaque-forming units/cell for 20 h. Cells were washed, refed, allowed to recover for 24 h, and then exposed to H2O2.
Generation and screening of stable cell lines.
The plasmid carrying the cDNA for CAT with a mitochondrial localization sequence was constructed as previously described (12, 24). MLE12 cells were transfected with lipofectamine plus reagent (Invitrogen, Carlsbad, CA). Stable cell lines were initially screened based on resistance to the appropriate antibiotics as previously described (3, 17). At least 15 colonies were isolated and screened for enzymatic activity.
Enzymatic activity of CAT was measured spectrophotometrically, as previously described (1). One unit of CAT activity was defined as the amount of enzyme required to decompose 1 μmol of H2O2 in 1 min at 25°C, pH 7.0.
Assay for mitochondrial function.
Mitochondrial activity was determined using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay to examine changes in mitochondrial dehydrogenase activity. Cells grown on six-well plates in 2 ml medium were exposed to 250 μM H2O2 for 1 h. MTT solution (200 μl, 5 mg/ml; Sigma) was added to each well, and the incubation was continued for another 1 h. Floating cells were spun down and combined with attached cells, and dye crystals were then dissolved in 2 ml isoproponol. The absorbance at 550 nm was determined on a 200-μl aliquot.
Assay for mitochondrial integrity (subcellular localization of cyt c).
Posttransduction (24 h), cells were seeded on a cover slip at the concentration of 50,000/well on a six-well plate, allowed to adhere overnight, and then treated with 250 μM H2O2 for 5 h. Cells in the supernatant were centrifuged on a slide at 500 revolutions/min for 2 min with a cytospin (Shandon, Pittsburgh, PA). Cells were fixed in 10% formalin buffered in PBS and permeablized with cold methanol. Cover slips and slides were then incubated with 1% BSA solution for 30 min with an anti-cyt c antibody (1:250 dilution; Calbiochem, San Diego, CA) for 2 h and washed with PBS. An FITC-conjugated secondary antibody (1:400 dilution; Calbiochem) was then applied for 1 h. The cover slips and slides were washed with excess water and incubated with Hoechst 33258 dye for 5 min. Cells were examined with a Nikon Diaphot 300 equipped with appropriate filters.
Subcellular localization of CAT.
Control cells were grown on cover slips and incubated with 10 nM of the mitochondrial dye MitoTracker Red (Molecular Probes, Eugene, OR) for 30 min. Cells were fixed and permeabilized as described above. Cover slips were then incubated with 1% BSA solution for 30 min, with an anti-CAT antibody (1:250 dilution; Calbiochem) for 30 min, and washed with PBS. An FITC-conjugated secondary antibody (1:50 dilution; Calbiochem) was then applied for 30 min. The cover slips were washed with excess water and examined on a Nikon Diaphot 300 equipped with appropriate filters (see below).
Mitochondrial respiration with MitoTracker Red CMH2Xros.
Transduced cells were seeded on a cover slip at a concentration of 50,000/well on a six-well plate. Postseeding (24 h), cells were treated with 250 μM H2O2 for 2 h. For the last 1 h of H2O2, cells were incubated with 500 nM MitoTracker Red CMH2Xros (Molecular Probes). Cells then were washed with 1× PBS and fixed in 10% formalin buffered in PBS for 15 min at room temperature.
All procedures were performed as described (17). The primary antibody was a phosphospecific JNK antibody (Cell Signaling, Beverly, MA) diluted 1:1,000. Secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit IgG (Cell Signaling) diluted 1:1,000. The immunoreactive proteins were visualized using the enhanced chemiluminescence solutions (Cell Signaling) on an IVIS50 Imaging System (Xenogen, Alameda, CA).
Image processing and analysis.
Mitochondrial localization was performed using computer-assisted image analysis. Images were acquired on a workstation consisting of a Nikon Diaphot 300 microscope, a Sutter filter wheel equipped with the appropriate filters, a Prior automated stage, a MicroMax 5 MHz Interline CCD camera (Roper Scientific), and the Metamorph imaging suite (Universal Imaging, West Chester, PA) under identical settings. For analysis of multiple fields, random fields of cells prepared for immunocytochemistry were captured, and colocalization and intensity of signal were determined with the Metamorph imaging software. Eighty to 100 cells per treatment group were analyzed and downloaded into Microsoft Excel for statistical analysis. For detailed Z-section analysis, ∼0.2-μm optical sections were acquired per field, and deconvolusion was performed using the Autodeblur and Autovisualize software package (AutoQuant Imaging, Watervliet, NY) with 20 iterations. The optical section with the mitochondria in the focal plane was selected for representation.
All data are reported as means ± SD. Statistical analysis was performed using Student's t-test or ANOVA with Fisher's protected least significant difference post hoc analysis using StatView version 5.01 (SAS Institute, Cary, NC). Pearson correlation testing was performed to analyze cell survival in terms of subcellular localization of CAT. P values <0.05 were considered significant.
Increased CAT expression and activity in viral transduced cells.
Replication-deficient type 5 adenovirus served as a viral vector to carry human CAT cDNAs with [mitochondrial CAT (MCAT)] and without [cytosolic CAT (CCAT)] an MnSOD mitochondrial localization sequence. A similar viral vector carrying β-galactosidase (LacZ) was used as a control for all experiments. Our previous results suggested that a two- to threefold increase in SOD enzymatic activity provided maximum protection against oxidant injury (14, 15), so an appropriate MOI was used to increase the activity into that target range. Increased enzymatic activity persisted for at least 1 wk and did not affect growth rate (data not shown). On average, an MOI of 100–125 resulted in a 2.1-fold increase of CAT activity in CCAT and 1.7-fold in MCAT-transduced cells relative to LacZ control cells. Minimal changes in SOD activity (CuZnSOD and MnSOD) were observed in all CCAT- and MCAT-transduced cells (compared with LacZ controls) after viral transduction (data not shown).
Mitochondrial localization of exogenous CAT in transduced cells.
Dual-labeling experiments were performed on viral-transduced cells to determine subcellular distribution of CAT. A diffuse, low-intensity staining pattern was observed in control (LacZ) cells, corresponding to a normal cytosolic distribution of CAT (Fig. 1A). Increased staining was observed in both CCAT- and MCAT-transduced cells, with a diffuse pattern in CCAT and a punctate distribution pattern in MCAT cells. The distribution pattern in MCAT cells suggested that expression was primarily mitochondrial in origin. The extent of mitochondrial expression was further assessed by counterstaining with Mitotracker, Z-axis analyses, and overlaying the resulting images. Mitochondrial expression of CAT was abundant in MCAT cells (orange/yellow regions) and was minimal in CCAT cells (Fig. 1B).
Mitochondrial localization provides better protection against H2O2-induced cell death.
To compare the ability of mitochondrial and cytosolic overexpression of CAT to protect against H2O2, cells were exposed to 250 μM H2O2. After exposure (5 h), there were 36% viable cells in LacZ control, 53% in CCAT, and 78% in MCAT cells (P < 0.05) compared with untreated ones. Although overexpression of CCAT provided better survival than controls (P < 0.01), overexpression of MCAT increased viability significantly compared with CCAT (P < 0.02; Fig. 2). These results demonstrate that overexpression of CAT in the mitochondria provides better protection against ROS-induced cell death compared with cytosolic localization. Improvements in cell survival were found in cells overexpressing MCAT compared with CCAT in A549 cells, a human lung epithelial cell line, suggesting that the advantages of mitochondrial localization are not limited to this particular cell line (data not shown).
To further investigate how mitochondrial localization of CAT affects viability, stable transfected clones overexpressing CAT specifically targeted to mitochondria were generated. Three clonal isolates that had varying increases in CAT activity (Table 1) and similar growth rates to parental controls were chosen for further examination. As shown in Fig. 3, the distribution of CAT closely resembles the mitochondrial distribution pattern in the MCAT1 clone, indicating localization of CAT primarily to mitochondria. Mitochondrial expression of CAT progressively decreased in clones MCAT5 and MCAT8. Ninety-eight percent of CAT localized to mitochondria in MCAT1, 70% in MCAT5, and only 30% in MCAT8 (Table 1). Cells were then exposed to 250 μM H2O2 for 5 h. Figure 4 demonstrates that, after H2O2 treatment, only 23% of parental cells were viable. In sharp contrast, all three mitochondrial overexpressors showed markedly improved cell survival. There was 140% survival in MCAT1 (with the highest MCAT activity), 90% survival in MCAT5, and 63% in MCAT8. Although MCAT8 had the highest total CAT activity (222%), this clone had the lowest mitochondrial localization (30%) and consequently the poorest survival among the three clones after H2O2 exposure. Pearson correlation analysis revealed that cell survival directly correlated with mitochondrial localization of CAT among the three clones (r = 0.60572, P < 0.05), rather than their overall enzymatic activity (r = −0.45501, P < 0.05).
To determine if overexpression of any protein in mitochondria could act as a nonspecific scavenger of H2O2, MLE12 cells were transduced with rAd.MnSOD (a mitochondrial enzyme). Despite significant improvement in cell survival in 95% O2 (14, 15), MnSOD failed to protect cells from H2O2-induced cell death (data not shown).
Mitochondrial localization of CAT improves mitochondrial integrity and function.
Mitochondria are primarily responsible for energy production in the cell. Damage by ROS not only disrupts these processes but also initiates apoptosis. We next investigated if cell survival directly correlated with preserved mitochondrial integrity and function. Cyt c is an enzyme located primarily in the mitochondrial intermembrane space. Disruption of mitochondrial integrity leads to cyt c release from mitochondria into the cytosol. Immunocytochemistry with an anti-cyt c antibody resulted in a typical punctate pattern in all cells before H2O2 treatment (Fig. 5, top). A 5-h exposure to H2O2 resulted in loss of cyt c staining in MLE12 mock (data not shown)- and LacZ-transduced cells and the presence of pycnotic nuclei (Fig. 5, bottom left), suggesting that cyt c is released in the cytosol. CCAT confers some protection from oxidant-mediated mitochondrial injury as seen by an intermediate level of intensity and punctuated pattern compared with untreated cells (Fig. 5, bottom middle). Less change in staining intensity or distribution pattern was observed in MCAT-transduced cells after treatment, indicating that mitochondrial localization of CAT maintains mitochondrial integrity (Fig. 5, bottom right). Mitochondrial dehydrogenase activity (a measure of mitochondrial function) was also examined. After exposure to H2O2 treatment (1 h), MTT values decreased to 31% in LacZ controls, 49.5% in CCAT, and 61.4% in MCAT. Thus overexpression of CAT in mitochondria optimally preserves function (2-fold compared with Lac Z controls), whereas cytosolic overexpression provides intermediate levels of protection (1.6-fold compared with LacZ controls; Fig. 6). Similar results were obtained in A549 cells where mitochondrial localization improved MTT activity 1.98 ± 0.01-fold (P < 0.001) and cytoplasmic localization 1.50 ± 0.01-fold (P < 0.02) compared with LacZ controls.
To further assess the extent of mitochondrial protection from oxidant damage, we utilized MitoTracker Red CMH2Xros, a redox-sensitive dye that fluoresces only in actively respiring mitochondria. As shown in Fig. 7, there is a higher level of fluorescence intensity in MCAT than in CCAT overexpressors both before and after H2O2 exposure. Thus mitochondrial localization of CAT preserves cell morphology, mitochondrial integrity, and mitochondrial function.
JNK pathway is inhibited in MCAT cells.
Our previous studies demonstrated that reducing JNK activity protected MLE12 cells from H2O2-induced cell death (17). Western blotting with antibodies recognizing the phosphorylated form of JNK revealed that the level of phosphorylated JNK significantly increased 30 min after exposure to H2O2 in LacZ-transduced MLE12 cells (Fig. 8). Exogenous expression of CAT was reduced significantly, with the lowest levels found in MCAT cells (Fig. 8). This demonstrates that CAT-mediated protection is, in part, through the JNK signaling pathway.
Mitochondrial localization of CAT also provides better protection in primary type II lung cells.
To validate our finding in primary cells, rat type II lung epithelial cells were isolated and transduced with CCAT and MCAT, along with LacZ as a control. Although incubation of cells with 250 μM of H2O2 for 1.5 h resulted in a significant cell death in LacZ control cells, cells overexpressing CCAT or MCAT showed little cell death (data not shown). Cells overexpressing MCAT had less cell death than CCAT cells after 1 mM of H2O2 treatment for 1.5 h (Fig. 9A), indicating that mitochondrial localization of CAT provided optimal protection against H2O2. Western blotting revealed that the level of phosphorylated JNK was reduced significantly in both CCAT- and MCAT-transduced cells compared with LacZ controls 30 min after exposure to H2O2 (data not shown), with levels in MCAT cells further reduced compared with CCAT cells (Fig. 9B).
The present study demonstrates that overexpression of CAT either in the cytosol or in mitochondria protects both transformed and primary lung epithelial cells from H2O2-induced apoptosis, although mitochondrial targeting provided significantly greater protection. Of particular importance, JNK activation after exposure to H2O2 was optimally inhibited in MCAT cells. Mitochondrial integrity (cyt c release) and function (dehydrogenase activity and mitochondrial respiration) was improved in MCAT cells as well. The degree of protection from oxidant injury directly correlated with the location of the CAT and not the overall increase in CAT activity.
Mitochondria are major sources of ROS production. Under conditions such as infection, stress, and hyperoxia, excess ROS will either be directly generated in mitochondria or diffuse into mitochondria. It is critical to scavenge ROS and prevent mitochondrial injury, which is the first step in the process of some forms of apoptotic cell death. Nomura et al. (23) demonstrated that overexpression of mitochondrial phospholipid hydroperoxide glutathione peroxidase (PHGPx) suppressed apoptosis mediated by exposure to oxidative stress, whereas overexpression of nonmitochondrial PHGPx failed to do so. Data from the present study demonstrate that mitochondrial structure and function can be preserved significantly and H2O2-induced apoptosis significantly ameliorated by targeting CAT, an AOE of cytosolic origin, to the mitochondria (Figs. 2 and 4–7). In agreement with our observations, a recent study examined a new class of antioxidants containing the triphenylphosphonium cation moiety that facilitates drug accumulation in mitochondria (9). When antioxidants such as Vit-E and ubiqinone are attached to triphenylphosphonium, these mitochondrial-targeted antioxidants preserve mitochondrial structure and function and prevent apoptosis caused by H2O2 significantly better than nontargeted therapies. Previous studies in Hep G2 cells demonstrated better protection from H2O2-induced apoptosis in cells overexpressing MCAT compared with cytosolic overexpressors despite similar protection between the two cell lines over a short period of time (3).
Despite higher overall enzymatic activity in cytosolic overexpressors, improvements in cell survival were intermediate (Fig. 2), suggesting that a critical level of mitochondrial AOE activity is required to protect mitochondria, as indicated by the mitochondrial function assays (Figs. 6 and 7). Overexpression of CAT in the cytosol protects cells in part by reducing the amount of H2O2 reaching the mitochondria, but diffusion of H2O2 into mitochondria or mitochondrial generation may be too fast to be effectively scavenged by CAT in the cytosol. If this occurs, the glutathione redox cycle system represents the only available protection against H2O2-induced injury in mitochondria. In a variety of conditions such as respiratory distress syndrome in adults (ARDS) and in premature neonates (RDS), glutathione levels in the lung may be reduced, and supplementation with n-acetylcysteine has not been shown to be efficacious in reducing lung damage from ROS (2).
Although sensitivity of cells to oxidant stress is related to cell cycle (i.e., cells with a longer cycle should be less vulnerable to oxidative stress), all transduced cells (LacZ, CCAT, and MCAT) had similar growth rates (data not shown). Therefore, it does not appear that differences in cell cycle characteristics played a significant role in these studies. In addition, cell growth is negligible during the relatively short time frame of these experiments with these doses of H2O2. The observation that stable MCAT1 cells continued to grow in H2O2 suggests that cell death is inhibited rather than delayed by overexpression of MCAT.
It is also possible that overexpression of any protein with cysteine residues in mitochondria could act as a nonspecific ROS scavenger. The fact that overexpression of mitochondrial MnSOD did not provide any protection against H2O2-induced injury demonstrates that protection offered by MCAT is specific and not the result of increased amounts of nonspecific protein in mitochondria.
Although both stable transfection and transient transduction improved cell survival, viral-mediated transduction was not as efficacious. It is likely due to the limitation of viral transduction efficiency where the CAT activity varies from cell to cell. In contrast, all clonally selected cells express the equivalent amount of CAT. It is interesting to note that survival in LacZ control cells was higher than parental MLE12 cells, presumably because of the cellular effects of viral infection, which upregulate some endogenous AOE. In fact, increased CAT activity was detected in LacZ-transduced cells compared with parental cells (data not shown). We have previously demonstrated that a two- to threefold increase in AOE activity is required to achieve optimal protection against various oxidant insults in MLE12 cells (14, 15). Higher AOE activity (>4- to 5-fold increase) is not as efficacious, although lower activity offers limited antioxidant protection (15). What is clear from the present studies is that cells with the highest overall CAT activity did not survive as well as cells with lower overall activity that localized primarily in mitochondria. The potential role of ROS (such as H2O2) as a second messenger in mediating growth-signaling pathways has been documented (20, 28), and drastic reductions in ROS production may impair normal cellular function. This has important clinical implications in the development of therapeutic interventions using AOE to prevent ROS-induced lung injury in critically ill patients.
The JNK kinase pathway is one of the leading candidates to transmit and transduce oxidant stress signaling into apoptosis signaling in various cell types, including lung epithelial cells (8, 13, 17, 18, 27). Studies from other laboratories have shown that activation of the JNK signaling pathway was sufficient to induce rapid cyt c release and mitochondrial-mediated apoptosis (16, 21, 32). We have previously demonstrated that inhibition of the JNK pathway by using dominant-negative JNK protected cells from H2O2-induced death (17). Our current data with both transformed lung epithelial cells and primary type II lung cells also indicate that overexpression of CAT prevents JNK activation and the downstream apoptosis pathway by scavenging ROS. This is consistent with findings from a recent publication that indicates that downregulation of the JNK pathway by overexpression of CAT in human arterial endothelial cells prevents oxidized low density lipoprotein-induced apoptosis (19). The fact that MCAT is more efficient in preventing upregulation of JNK suggests that JNK is important in mediating cellular and mitochondria responses to ROS and once more emphasizes the importance of mitochondria in ROS-induced cell death. In agreement with our notion, a recent study demonstrated that specifically overexpressing human DNA repair enzymes in mitochondria prevented apoptosis induced by ROS in human lung endothelial cells (25). It is important to note that the reduction in JNK activation between cells overexpressing CCAT or MCAT is not as significant in primary type II cells compared with cultured MLE12 cells. This may be explained by 1) transduction efficiency was not high enough to exhibit significant difference, and 2) primary cells behave differently from cultured MLE12 cells. Despite this, inhibition of JNK activation does occur in primary cells overexpressing CAT. The levels of ROS in cells overexpressing CAT in mitochondria and the relation to JNK activation and reduced cell death in H2O2 are currently under further investigation.
Alternative methods of AOE administration have been proposed to maximize efficacy and minimize toxicity, such as vascular immunotargeting using anti-platelet endothelial cell adhesion molecule/AOE complexes that can localize rapidly and primarily to the pulmonary vasculature and prevent acute lung injury caused by H2O2 (6). It would not be difficult to add a mitochondrial targeting sequence to this complex and, in theory, improve the efficacy of this treatment. Our ultimate goal is to develop new therapies for the prevention and treatment of ROS-induced lung injury in neonates, children, and adults. Cell and site-specific delivery of AOE and derivatives in optimal concentrations may maximally protect the lung from oxidant injury and lead to novel therapies for the prevention of ROS-induced lung damage.
This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-64158 (J. M. Davis and J. A. Kazzaz) and HL-49959 (A. Chander), Philip Morris USA, and Philip Morris International.
We thank Dr. S. Pollack for assistance with statistical analyses. We also thank Delon Callender for the type II cell preparation.
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.
- Copyright © 2006 the American Physiological Society