Mitochondrial Complex III-generated Oxidants Activate ASK1 and JNK to Induce Alveolar Epithelial Cell Death following Exposure to Particulate Matter Air Pollution*

We have previously reported that airborne particulate matter air pollution (PM) activates the intrinsic apoptotic pathway in alveolar epithelial cells through a pathway that requires the mitochondrial generation of reactive oxygen species (ROS) and the activation of p53. We sought to examine the source of mitochondrial oxidant production and the molecular links between ROS generation and the activation of p53 in response to PM exposure. Using a mitochondrially targeted ratiometric sensor (Ro-GFP) in cells lacking mitochondrial DNA (ρ0 cells) and cells stably expressing a small hairpin RNA directed against the Rieske iron-sulfur protein, we show that site III of the mitochondrial electron transport chain is primarily responsible for fine PM (PM2.5)-induced oxidant production. In alveolar epithelial cells, the overexpression of SOD1 prevented the PM2.5-induced ROS generation from the mitochondria and prevented cell death. Infection of mice with an adenovirus encoding SOD1 prevented the PM2.5-induced death of alveolar epithelial cells and the associated increase in alveolar-capillary permeability. Treatment with PM2.5 resulted in the ROS-mediated activation of the oxidant-sensitive kinase ASK1 and its downstream kinase JNK. Murine embryonic fibroblasts from ASK1 knock-out mice, alveolar epithelial cells transfected with dominant negative constructs against ASK1, and pharmacologic inhibition of JNK with SP600125 (25 μm) prevented the PM2.5-induced phosphorylation of p53 and cell death. We conclude that particulate matter air pollution induces the generation of ROS primarily from site III of the mitochondrial electron transport chain and that these ROS activate the intrinsic apoptotic pathway through ASK1, JNK, and p53.

We have previously reported that airborne particulate matter air pollution (PM) activates the intrinsic apoptotic pathway in alveolar epithelial cells through a pathway that requires the mitochondrial generation of reactive oxygen species (ROS) and the activation of p53. We sought to examine the source of mitochondrial oxidant production and the molecular links between ROS generation and the activation of p53 in response to PM exposure. Using a mitochondrially targeted ratiometric sensor (Ro-GFP) in cells lacking mitochondrial DNA ( 0 cells) and cells stably expressing a small hairpin RNA directed against the Rieske iron-sulfur protein, we show that site III of the mitochondrial electron transport chain is primarily responsible for fine PM (PM 2.5 )-induced oxidant production. In alveolar epithelial cells, the overexpression of SOD1 prevented the PM 2.5 -induced ROS generation from the mitochondria and prevented cell death. Infection of mice with an adenovirus encoding SOD1 prevented the PM 2.5 -induced death of alveolar epithelial cells and the associated increase in alveolar-capillary permeability. Treatment with PM 2.5 resulted in the ROS-mediated activation of the oxidant-sensitive kinase ASK1 and its downstream kinase JNK. Murine embryonic fibroblasts from ASK1 knock-out mice, alveolar epithelial cells transfected with dominant negative constructs against ASK1, and pharmacologic inhibition of JNK with SP600125 (25 M) prevented the PM 2.5 -induced phosphorylation of p53 and cell death. We conclude that particulate matter air pollution induces the generation of ROS primarily from site III of the mitochondrial electron transport chain and that these ROS activate the intrinsic apoptotic pathway through ASK1, JNK, and p53.
Epidemiologic studies have consistently demonstrated a strong link between the daily levels of particulate matter air pollution Ͻ2.5 m in diameter (PM 2.5 ) 3 and PM Ͻ10 m in diameter (PM 10 ) and cardiopulmonary morbidity and mortality (1)(2)(3). In humans, exposure to PM 10 has been associated with an increase in mortality from ischemic cardiovascular events including stroke and myocardial infarction, an acceleration in the age-related decline in lung function in normal adults, impairment in normal lung development in children, exacerbations of asthma in children and adults, accelerated atherosclerosis in women, increased rates of lung cancer, and the development of myocardial ischemia in men with stable coronary artery disease (4 -10). The intracellular generation of reactive oxygen species (ROS) has emerged as a common mechanism by which particulates might initiate signaling pathways that end in these diverse pathologic conditions (11). We have reported that the PM-induced generation of ROS requires a functional electron transport chain, suggesting that PM might induce the inadvertent transfer of electrons from one or more sites in the electron transport chain to molecular oxygen (12).
One of the mechanisms by which exposure to PM can contribute to alveolar epithelial dysfunction, lung injury and inflammation, and lung cancer is by activating the intrinsic apoptotic pathway to induce cell death (11,12). We have reported that this process requires the activation of p53; however, the molecular events linking the generation of ROS by the mitochondrial electron transport chain with the activation of p53 are not known (12). In this paper, we show that exposure of alveolar epithelial cells to PM 2.5 induces the generation of ROS from site III of the mitochondrial electron transport chain. These mitochondrially derived oxidants activate the mitogenactivated signaling kinase kinase kinase (MAPKKK) apoptosis signaling kinase 1 (ASK1), which activates the c-Jun N-terminal kinase (JNK) signaling pathway. The activation of JNK is required for the phosphorylation of p53 and the subsequent cell death. Inhibition of mitochondrial oxidant production in mouse lungs prevents PM 2.5 -induced cell death and the associ-ated PM 2.5 -induced increase in the permeability of the alveolarcapillary barrier.

EXPERIMENTAL PROCEDURES
Particulate Matter (PM 2.5 )-The Washington, D. C. ambient PM 2.5 was obtained from the National Institutes of Standards and Technology (SRM 1649a) (13). The characteristics of the PM 2.5 have been described previously (14).
Isolation and Culture of Alveolar Epithelial Cells-Alveolar type II cells were isolated from rats as described previously (15) and used 2 days after isolation. A549 cells were obtained from ATCC. All of the cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum with 2 mM L-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin. The 0 A549 cells were generated by incubating wild type cells in medium containing ethidium bromide (100 ng/ml), sodium pyruvate (1 mM), and uridine (100 g/ml) for 3-5 weeks. The lack of DNA encoding cytochrome oxidase subunit IV was confirmed by PCR using the primer sequences described previously (16).
Plasmids, Adenoviral, Lentiviral, and Retroviral Vectors-An adenoviral vector encoding Ro-GFP with a mitochondrial localization sequence (mito-Ro-GFP) was generated as described previously and commercially amplified (ViraQuest, Iowa City, IA) (17). Adenoviral vectors encoding SOD1 and SOD2 were kind gifts of Dr. John Engelhardt and were purchased from the University of Iowa viral vector core (18,19). The retrovirus encoding a shRNA for the Rieske iron-sulfur protein was generated as described previously (20) and amplified in Platinum E packaging cells. The packaging cells and the cells infected with the Rieske iron-sulfur shRNA were grown in medium containing 100 g/ml uridine. A lentiviral vector encoding the mito-Ro-GFP probe with a mitochondrial localization sequence was created using the ViraPower lentiviral transformation kit according to the manufacturer's directions (Invitrogen). The PCR product from the plasmid sequence was amplified using the following primers: forward, 5Ј-CAC CAT GCC GCT AGC GCC-3Ј, and reverse, 5Ј-TTA CTT GTA CAG CTC GTC-3Ј; then the PCR product was cloned into the ViraPower lentiviral plasmid and transfected into packaging cells, and the filtered supernatant was used to infect A549 cells. The cells were selected by their ability to grow in blasticidin (5 g/ml) and/or puromycin (5 g/ml). Transient transfection of A549 cells was performed by Lipofectamine 2000 according to the manufacturer's directions (Invitrogen).
Measurement of Reactive Oxygen Species-We employed an oxidant-sensitive ratiometric probe (Ro-GFP) that was originally described by Remington and co-workers (21,22), who validated its responsiveness to a variety of intracellular oxidants both ex vivo and in living cells. The sequence for the Ro-GFP probe with a mitochondrial localization sequence was cloned into a lentiviral vector (Virapower Lentiviral Expression System; Invitrogen) and was expressed in packaging cells. The resulting virus was used to infect A549 cells, and stably expressing clones were selected by their ability to grow in blasticidin. The vector was also cloned into an adenoviral vector as described previously. Expression of the probe was confirmed by examining the cells using fluorescence microscopy. Appropriate localization of the probe to the mitochondria was confirmed by co-staining with the mitochondrial probe MitoTracker (10 M, 10 min in the dark) (Invitrogen).
Oxidation of the mito-Ro-GFP probe was assessed using flow cytometry. After treatment, the cells were removed from the plate using trypsin, and equal aliquots of the resulting suspension were transferred to tubes containing medium alone or medium containing 1 mM dithiothreitol or 1 mM t-butyl hydroperoxide (t-BOOH). After 10 min, the ratio of fluorescence (emission of 535 nm) at excitations of 405 and 488 nm was measured in 5,000 cells/condition using a DakoCytomation CyAn high speed multilaser droplet cell sorter. The oxidation state of the cells was calculated as the completely reduced ratio (dithiothreitol) less the untreated value divided by the difference in the ratio observed with dithiothreitol and t-BOOH (17).
Animals and Intratracheal Administration of Particulate Matter-The protocol for the use of mice was approved by the Animal Care and Use Committee at Northwestern University. 6 -8-week-old, (20 -25 g) male C57BL/6 mice (Charles River) were anesthetized with pentobarbital (50 mg/kg intraperitoneally) and intubated orally with a 20-gauge angiocath cut to a length that placed the tip above the carina (23,24). We instilled either PM 2.5 suspended in 50 l of sterile PBS or 50 l of sterile PBS (control) in two equal aliquots, 3 min apart. Particulate matter was vortexed immediately prior to instillation. After each aliquot the mice were placed in the right and then the left lateral decubitus positions for 10 -15 s. Adenoviral Infection in Mice-Type I adenoviral vectors were instilled into the lungs of mice as described previously (25). Briefly, the mice were anesthetized with sodium pentobarbital or isoflurane and intubated with a 20-gauge angiocath. A Hamilton syringe was used to instill 1 ϫ 10 9 pfu of virus in 50% bovine surfactant (Infrasurf; Forest Pharmaceuticals) and balance Tris-EDTA buffer through the angiocath. The virus was administered in two equal aliquots, 3-5 min apart, after which the animals were extubated and allowed to recover from anesthesia with the administration of supplemental oxygen as required to treat hypoventilation.
Histology-A 20-gauge angiocath was sutured into the trachea, the lungs and heart were removed en bloc, and the lungs were inflated to 15 cm of H 2 O with 4% paraformaldehyde. The heart and lungs were fixed in paraffin, and 5-m sections were stained with hematoxylin/eosin (15).
Assessment of Lung Permeability-Lung permeability was assessed using a modification of a described previously tech-nique (26). The mice were anesthetized with pentobarbital, and 125 l of fluorescein isothiocynate-dextran (molecular mass, 4,000 kDa) 0.05 g/ml (Sigma-Aldrich) was delivered into the retro-orbital plexus using a 26-gauge needle. The mice were kept sedated for 30 min, after which a 20-gauge angiocath was sutured into the trachea for the collection of bronchoalveolar lavage fluid. Immediately following the bronchoalveolar lavage, 450 l of blood was collected from the right ventricle into a syringe containing 50 l of sodium citrate (final dilution 1:10). Relative lung permeability was estimated from the difference in the fluorescence of the plasma and the bronchoalveolar lavage fluid measured by using a microplate reader (excitation, 490 nm; emission, 520 nm).
Assessment of Cell Death-Cell death was assessed using a commercially available photometric immunoassay that detects histone-associated DNA fragments (Roche Applied Science); an antibody that detects cleaved caspase-3 in cell lysates and a commercially available assay that detects caspase-9 activity (BioVision, Mountain View, CA).
Immunoblotting-Protein immunoblotting was performed as described previously (26). The cell lysates were mixed with sample loading buffer (125 mM Tris base (pH 6.8), 4% (w/v) SDS, 20% (v/v) glycerol, 200 mM dithiothreitol, 0.02% (w/v) bromphenol blue). After heating, the protein was resolved on a SDS-15% polyacrylamide gel and transferred to a Hybond-ECL nitrocellulose membrane (Amersham Biosciences). After transfer, the gel was stained with Ponceau S to verify uniform transfer. The membranes were blocked with 5% (w/v) nonfat milk in TBS-T (100 mM Tris base, pH 7.5, 0.9% (w/v) NaCl, 0.1% (v/v) Tween 20) for 2 h at room temperature and subsequently incubated with the appropriate primary antibody overnight at 4°C. The membrane was washed with TBS-T three times and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody. The membrane was washed three times with TBS-T and analyzed by enhanced chemiluminescence (Amersham Biosciences). Relative quantification was performed using the ratio of the densitometry of the protein of interest to the appropriate loading control as measured using Image J software (16).
Kinase Assays to Measure the Activity of c-Jun-Kinase assays to measure the activity of c-Jun were performed using a commercially available assay according to the manufacturer's directions (Cell Signaling; catalog number 9810), except the incubation with ATP was extended to 1 h. Kinase assays were also performed using a modification of a procedure described previously (27). Briefly, cell lysates were obtained after exposure to PM 2.5 , and c-Jun was immunoprecipitated by incubating ϳ500 g of protein with 5 l of c-Jun antibody overnight (4°C) (Santa Cruz Biotechnology Inc.). The complexes were pulled down with 50 l of protein A/G-agarose beads and then washed in lysis buffer. The immunoprecipitates were incubated with 2 g of myelin basic protein, 10 Ci of [␥-32 P]ATP, 100 M ATP for 30 min at 30°C in 35 mM Tris, pH 7.5, 10 mM MgCl 2 , 5 mM EGTA, 1 mM CaCl 2 , and 10 mM ␤-glycerolphosphate. The reactions were stopped by adding SDS sample buffer and boiling the samples. The proteins were separated by SDS-PAGE, and radiolabeled c-Jun was detected by autoradiography.
TUNEL Staining of Lung Sections-End labeling of exposed 3Ј-OH ends of DNA fragments in paraffin-embedded tissue was done utilizing the TUNEL AP in situ cell death detection kit (Roche Applied Science) according to manufacturer's directions. After staining, 20 fields of alveoli (400ϫ) were randomly chosen, and the nuclei and total nuclei were counted using an automated program (Image J) (26).
Statistics-Differences between groups were explored using analysis of variance. When the analysis of variance indicated a significant difference, individual differences were explored using t tests with a Dunnett correction for multiple comparisons against control conditions. All of the analyses were performed using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA).

The Generation of ROS Is Required for PM 2.5 -induced Cell
Death in Alveolar Epithelial Cells-We previously reported that exposure of alveolar epithelial cells to PM 10 collected from Düsseldorf, Germany induced the generation of ROS (15). To determine whether PM 2.5 -induced cell death is oxidantdependent, we infected primary rat alveolar type II cells and A549 cells with an adenoviral vector encoding mito-Ro-GFP 48 h before exposure to PM 2.5 (50 g/cm 2 ) from Washington, D. C. Four hours later, we measured the oxidation of the probe in cells that had been treated with PBS vehicle or with the combined superoxide dismutase and catalase mimetic EUK-134 (20 M) for 30 min before and during the PM 2.5 exposure (28). Treatment with EUK-134 prevented the PM 2.5 -induced oxidation of the probe in both A549 cells and primary alveolar type II cells. Treatment with t-butyl hydroperoxide was used as a positive control (Fig. 1A). Cell death (DNA fragmentation) was measured in identically treated cells 24 h after PM 2.5 exposure. Treatment with EUK-134 prevented cell death in response to PM 2.5 in A549 and primary rat alveolar epithelial cells (Fig. 1B).
PM-induced Cell Death Requires the Generation of ROS by the Mitochondria in Alveolar Epithelial Cells-Using an oxidant-sensitive fluorescent dye, we previously reported that the administration of PM 10 to alveolar epithelial cells resulted in the generation of ROS from the mitochondria. To directly measure PM 2.5 -induced mitochondrial oxidant production, we generated A549 cells lacking mitochondrial DNA ( 0 A549 cells). Wild type and 0 A549 cells were infected with an adenovirus encoding the mito-Ro-GFP, and the oxidation of the probe was measured every 10 s after treatment with PM 2.5 (50 g/cm 2 ) or PBS using flow cytometry. Treatment with PM 2.5 resulted in rapid (60 s) oxidation of the probe in wild type cells; however, minimal oxidation of the probe was seen in 0 cells ( Fig. 2A).
Superoxide anion generated from site III of the mitochondrial electron transport chain can be released into the mitochondrial matrix where it is metabolized by SOD2, or it can be released into the intermembrane space where it is metabolized by SOD1 (29). The resulting H 2 O 2 is reduced by catalase or indirectly by glutathione peroxidase, both of which are present in excess in the mitochondria (29). The cells were simultaneously infected with the 2 pfu/cell of the mito-Ro-GFP virus and 5 pfu/cell of a null virus (Ad Null), SOD1 (Ad SOD1), or SOD2 (Ad SOD2). Twenty-four hours later the cells were treated with PM 2.5 (50 g/cm 2 ), and the oxida-tion of the probe was measured every 10 s for 250 s using flow cytometry. Compared with PBS-treated cells, the cells infected with the null virus showed significant oxidation of the probe after treatment with PM 2.5 (Fig. 2B). Cells infected with SOD1 showed increased expression of SOD1 in whole cell lysates and showed no significant oxidation of the probe in response to PM 2.5 . The cells infected with SOD2 showed increased expression of SOD2 in whole cell lysates and an attenuated PM 2.5 -induced oxidation of the probe (Fig. 2, C and D). Similar results were obtained when identically infected cells were exposed to PM 2.5 (50 g/cm 2 ), and oxidation of the probe was measured 4 h later (Fig. 2E).
To determine whether the inhibition of oxidant generation by SOD1 or SOD2 could prevent cell death, A549 cells were infected with a null virus, SOD1, or SOD2 24 h before exposure to PM 2.5 (50 g/cm 2 ) or PBS, and DNA fragmentation was measured 24 h later. Compared with cells infected with the null virus, there was a significant increase in DNA fragmentation in cells infected the SOD2 adenovirus 24 h after treatment with PM 2.5 . By contrast, no significant increase in DNA fragmentation was observed in cells infected with SOD1 (Fig. 3A). Consistent with these results, cells infected with the null or SOD2 adenovirus, but not the SOD1 adenovirus, showed a significant increase in caspase-9 activity (Fig. 3B) and cleavage of caspase-3 (Fig. 3C) 24 h after treatment with PM 2.5 .
Site III of the Mitochondrial Electron Transport Chain Is Required for PM 2.5 -induced ROS Generation-The bulk of mitochondrial oxidant production occurs in site I or III of the mitochondrial electron transport chain (29). At site III, the inadvertent transfer of a single electron to molecular oxygen during the ubiquinone/ubisemiquinone or Q cycle can generate a superoxide anion. To determine whether oxidant generation at site IIII of the mitochondrial electron transport chain was required for PM 2.5 -induced ROS production, we used a lentiviral vector to generate a stable A549 cell line expressing mito-Ro-GFP. This cell line was then infected with a retroviral vector expressing a shRNA against the Rieske iron-sulfur protein or with a control shRNA vector (Drosophila hypoxia-inducible factor). The Rieske iron-sulfur protein is required for the generation of the ubisemiquinone radical from ubiquinol; the loss of this protein effectively blocks the transfer of electrons through the Q cycle (29). The cells were selected by their growth in blasticidin (lentiviral vector) and puromycin (retroviral vector). The resulting cells had significantly reduced expression of the Rieske iron-sulfur protein (Fig. 4A) and required uridine for survival as assessed by trypan blue staining (not shown). These cells were then treated with PM 2.5 (50 g/cm 2 ), and the oxidation of the mito-Ro-GFP probe was measured 4 h later. Compared with wild type cells, the cells with a knockdown of the Rieske iron-sulfur protein had significantly less oxidation of the probe in response to PM 2.5 (Fig. 4B). Consistent with these findings, cell death (DNA fragmentation) was not different from untreated in cells lacking the Rieske ironsulfur protein 24 h after treatment with PM 2.5 (50 g/cm 2 ) (Fig. 4C).
The Overexpression of SOD1 in the Lung Prevents PM 2.5 -induced Cell Death and Alveolar Capillary Barrier Dysfunction in Mice-To determine whether ROS generation was required for PM 2.5 -induced cell death in vivo, we infected mice with a null adenovirus or an adenovirus encoding SOD1. Seven days after infection, the mice were intratracheally treated with 50 l of sterile PBS or PM 2.5 suspended in the same volume of PBS (200 g/animal). Compared with mice infected with the null adenovirus, the levels of SOD1 were significantly higher in lung homogenates obtained 7 days after infection from mice infected with the SOD1 adenovirus (Fig. 5A). The number of TUNEL-positive nuclei observed in lung sections obtained 24 h after treatment with PM 2.5 was higher in mice treated 7 days earlier with a null adenovirus than in mice treated with SOD1 (Fig. 5B). Furthermore, the increase in alveolar-capillary permeability observed in the null virus-infected animals 24 h after treatment with PM 2.5 was not observed in mice infected with the SOD1 adenovirus (Fig. 5C). Examination of the lungs of null and SOD1-infected animals treated with PM 2.5 24 h earlier  JANUARY 23, 2009 • VOLUME 284 • NUMBER 4

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revealed a modest inflammatory response that was not observed in mice treated with PBS (Fig. 5D). PM 2.5 -induced Cell Death Requires the Oxidant-mediated Activation of ASK1 and JNK-We then sought to examine the molecular link between mitochondrial oxidant production and p53-dependent alveolar epithelial cell death. The MAPKKK ASK-1 has been shown to be activated in response to oxidant stress and to activate cell death primarily through its ability to activate the JNK pathway. We treated A549 cells with PM 2.5 in the presence or absence of EUK-134 (20 M) or PBS and immunoblotted cell lysates using an antibody against phosphorylated ASK1. Treatment with PM 2.5 resulted in rapid activation of ASK1 (Fig. 6A) that was inhibited by pretreatment with EUK-134 (Fig. 6B). Cells lacking mitochondrial DNA ( 0 cells) failed to phosphorylate ASK1 in response to PM 2.5 (Fig. 6C). Transient transfection of A549 cells with a dominant negative ASK1 plasmid 24 h before the exposure prevented PM 2.5 -induced cell death (Fig. 6D). To confirm the importance of ASK1 in PM 2.5 -induced cell death, we exposed murine embryonic fibroblasts (MEFs) from wild type and ASK1 Ϫ/Ϫ knock-out mice to PM 2.5 and 24 h later measured cell death (DNA fragmentation). Wild type but not ASK1 Ϫ/Ϫ MEFs showed a dose-dependent increase in cell death in response to PM 2.5 (Fig. 6E). In other models of oxidant stress, the activation of ASK1 has been shown to exert its pro-apoptotic effects by activating the JNK pathway. To determine whether ASK1 was required for PM 2.5 -induced JNK activation, we measured the phosphorylation of c-Jun by lysates of PM-or PBS-treated wild type and ASK1 Ϫ/Ϫ MEFs using a commercially available JNK kinase assay. Activation of JNK was observed in wild type but not ASK1 Ϫ/Ϫ MEFs 4 h after exposure to PM 2.5 (Fig. 6E).
To further examine the role of JNK in PM 2.5 -induced cell death, we measured JNK phosphorylation in primary rat alveolar type II cells after treatment with PM 2.5 . Compared with PBS-treated cells, JNK phosphorylation was increased as early as 15 min after treatment with PM 2.5 (50 g/cm 2 ) and was maximal after 30 min (not shown). The phosphorylation of JNK observed in cells 30 min after treatment with PM 2.5 was prevented by treatment of the cells with EUK-134 (20 M) for 30 min before and during a 30-min exposure to PM 2.5 . (Fig. 7A). PM 2.5 -induced activation of the JNK pathway was confirmed in A549 cells using a standard kinase assay that measures the phosphorylation of c-Jun by cell lysates using radiolabeled phosphorus ( 32 P) in the presence or absence of the JNK inhib-

-induced mitochondrial oxidant production is inhibited by the overexpression of SOD1.
A, wild type and 0 A549 cells were infected with mito-Ro-GFP, and 24 h later the cells were treated with PM 2.5 (50 g/cm 2 ) or PBS. The cells were harvested at the times shown, and the oxidation state of the probe relative to the fully reduced and oxidized values was measured using flow cytometry. B-D, A549 cells were infected with an adenovirus encoding no transgene (Ad Null, B) or increasing amounts of an adenovirus encoding SOD1 (Ad SOD1, C) or SOD2 (Ad SOD2, D) measured in pfu, and 24 h later their abundance was measured in cell lysates by immunoblotting. Separate groups of cells were simultaneously infected with 2 pfu/cell of the mito-Ro-GFP virus and 5 pfu/cell of Ad Null, Ad SOD1, or Ad SOD2. Twenty-four hours later the cells were treated with PM 2.5 (50 g/cm 2 ), and the oxidation of the probe was measured every 10 s for 250 s using flow cytometry. E, cells infected as in B-D were treated PM 2.5 (5 g/cm 2 ), and oxidation of the probe was measured 4 h later using flow cytometry. An asterisk indicates p Ͻ 0.05 when compared with untreated controls. There are a minimum of three replicates/experiment. itor SP600125 (25 M) (Fig. 7B). A549 cells infected 24 h earlier with the null adenovirus, SOD1, or SOD2 were treated with PM 2.5 (50 g/cm 2 ), and 30 min later, the cell lysates were assessed for the activation of JNK using a commercially available kinase assay. Compared with the null virus-infected cells, the cells infected with SOD1 showed no significant activation of JNK1 in response to PM 2.5 , whereas cells infected with SOD2 showed an attenuated response (Fig. 7C). To determine whether oxidant generation at site III of the mitochondrial electron transport chain is required for the PM 2.5 -induced activation of JNK, we measured PM 2.5 -induced JNK activation in A549 cells stably transfected with a control retrovirus encoding Drosophila HIF or a virus encoding an shRNA to the Rieske iron-sulfur protein. Thirty minutes after treatment with PM 2.5 , robust activation of JNK was observed in control transfected but not Rieske iron-sulfur transfected cells (Fig. 7D). To determine whether activation of JNK was required for PM 2.5 -induced cell death, we treated primary alveolar type II cells with the JNK inhibitor SP600125 (25 M) 30 min before and during treatment with PM 2.5 , and cell death was assessed 24 h later (DNA fragmentation). The cells treated with SP600125 were protected against PM 2.5 -induced cell death (Fig. 7E). Similar protection was observed in A549 cells (data not shown). We have previously reported that exposure to PM increases the protein abundance and transcriptional activity of p53 and that the transcriptional activity of p53 is required for PM 10 -induced apoptosis in vitro and in vivo. In other models of oxidant stress, JNK-mediated phosphorylation of Ser-15 has been associated  . Site III of the mitochondrial electron transport chain is required for PM 2.5 -induced oxidant production and cell death. A, protein abundance of the Rieske iron-sulfur (Fe-S) protein was measured by immunoblotting in a stable line of A549 cells expressing the mito-Ro-GFP and a shRNA against the Rieske iron-sulfur protein or a control shRNA. B, these cells were treated with PM 2.5 (50 g/cm 2 ) or PBS, and 4 h later oxidation of the probe was measured using flow cytometry. C, identically treated cells were assayed for cell death (DNA fragmentation) 24 h after treatment. An asterisk indicates p Ͻ 0.05 when compared with untreated controls. There are a minimum of three replicates/experiment. with the stabilization and enhanced transcriptional activity of p53. We treated primary rat alveolar type II cells with SP600125 as described above and measured the phosphorylation of p53 at Ser-15 4 h later (Fig. 7F). Treatment with SP600125 prevented the PM 2.5 -induced phosphorylation of p53 at Ser-15.

DISCUSSION
Exposure to airborne particulate matter air pollution is associated with significant increases in cardiopulmonary morbidity and mortality (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). Although an increasing body of evidence has linked the production of ROS in response to the particles with their toxicity, less is known regarding the molecular mechanisms by which the particles generate oxidants and the link between these oxidants and intracellular signaling pathways. Our observation that knockdown of the Rieske iron-sulfur protein in lung epithelial cells prevents PM 2.5 -induced mitochondrial oxidant production suggests that site III of the mitochondrial electron transport chain is a major contributor to PM 2.5 -induced oxidant production. Our results suggest that the superoxide anion produced at site III is released into the mitochondrial intermembrane space and from there it signals to the cytosol to induce the activation of the oxidant-sensitive kinase ASK1. The activation of ASK1 is required for the PM 2.5 -induced activation of JNK, which phosphorylates p53 to induce cell death. The importance of oxidant-mediated apoptosis in the injury induced by PM 2.5 is highlighted by our observation that adenovirally mediated overexpression of SOD1 in the lung epithelium attenuates PM 2.5 -induced apoptosis and prevents the PM 2.5 -induced increase in the permeability of the alveolar-capillary barrier.
The mitochondrial electron transport chain is a multiprotein complex in the mitochondrial inner membrane that sequentially transfers electrons generated during glycolysis and the Krebs cycle through intermediate molecules to molecular oxygen. The free energy released in these reactions is used to generate the chemiosmotic gradient required for the synthesis of ATP. The electron transport chain is traditionally divided into four sites or complexes, each of which contains multiple proteins. Critical components of all four sites of the electron transport chain as well as components of the F 1 F 0 synthase are included in the 13 nontranscriptional proteins encoded by the mitochondrial genome. Sites I and II transfer electrons from reduced carriers (NADH or FADH) to site III, which contains the cytochrome bc 1 complex. Transfer of electrons from cytochrome c to molecular oxygen is accomplished by the cytochrome c oxidase (site IV). The transfer of electrons to the cytochrome bc 1 complex requires the sequential transfer of two electrons from the Rieske iron-sulfur protein to the ubiquinol/ ubisemiquinone or Q cycle. Ubisemiquinone, a radical, is generated twice during the cycle and is predisposed to transfer an electron to molecular oxygen producing a superoxide radical. Generation of ROS by the Q cycle is seen dramatically in cells in which cytochrome b is genetically absent or inhibited (e.g. after treatment with antimycin A). We observed that cells lacking mitochondrial DNA ( 0 cells) failed to generate ROS in response to PM 2.5 , suggesting that a functional electron transport chain was required for their generation. We generated a stable knockdown of the Rieske iron-sulfur protein to prevent the flow of electrons into the Q cycle. Our observation that these cells failed to generate ROS in response to PM 2.5 suggests that the Q cycle is responsible for the bulk of PM 2.5 -induced oxidant generation.
The ubisemiquinone radical is generated twice during a full cycle of the Q cycle, once on the outer surface of the membrane (the Q o site) and once closer to the inner surface of the membrane (the Q i site). Superoxide generated at the Q o site is released into the intermembrane space, whereas superoxide anion generated at the Q i site is released into the mitochondrial matrix. A dismutation reaction between the superoxide radicals can result in the generation of hydrogen peroxide and oxygen. This reaction is efficiently catalyzed by the enzyme super- oxide dismutase, SOD. The primary SOD in the mitochondrial matrix is SOD2, whereas SOD1 is primarily expressed in the mitochondrial intermembrane space and the cytosol. We observed that overexpression of SOD1, but not SOD2, prevented the PM 2.5 -induced generation of ROS. These results, combined with those we obtained using 0 cells and cells with a stable knockdown of the Rieske iron-sulfur protein, suggest that PM 2.5 -induced mitochondrial oxidant production originates from the Q o site. These results provide two lines of evidence against the generation of significant quantities of superoxide anion from Sites I or II of the electron transport chain in response to PM. First, in cells with a stable knockdown of the Rieske iron-sulfur protein, the electron carriers at site I should be fully reduced, increasing the tendency toward ROS production. Second, because the proteins comprising these complexes are primarily located in the mitochondrial matrix; superoxide anion generated at these sites would be expected to be dismutated by SOD2 (29).
Nel and co-workers (30) have carefully examined the constituents of PM 2.5 obtained in Los Angeles and their effects in isolated cells. The particles consist of a core of carbon or ash that is generated during the combustion of fossil fuels. After combustion, this core rapidly cools and organic materials, and transition metals present in the combustion chamber are absorbed onto the particle. Xia et al. (31,32) fractionated the organic component of the particles and demonstrated that the fraction enriched in quinones was sufficient to increase oxidant generation in an isolated liver mitochondria preparation. In A549 cells, Li et al. (33) showed the accumulation of electron dense granules 1 h after particle administration. Our observations extend these findings by localizing the site of oxidant generation to site III of the mitochondrial electron transport chain in the intact cell. The fact that we observed a very rapid (within minutes) oxidation of the mitochondrially localized probe following the addition of PM 2.5 makes it less likely that the uptake of the particles and their localization to the mitochondria are solely responsible for their oxidant generation.
We used an oxidant-sensitive GFP probe to detect the generation of reactive oxygen species in response to PM 2.5 . Hanson et al. (22) reported that these probes respond to the generation of both superoxide anion and H 2 O 2 with inverse changes in the emission from their excitation maxima near 400 and 490 nm. They further showed that the addition of targeting sequences A, A549 cells were exposed to PM 2.5 (50 g/cm 2 ) for the times indicated, and the phosphorylated ASK1 was measured by immunoblotting. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. B, cells were treated with EUK-134 (5 M) or vehicle (PBS) 30 min before and during 30 min of exposure to PM 2.5 , and phosphorylated ASK1 was measured by immunoblotting. C, wild type and 0 A549 cells were treated with PM 2.5 (50 g/cm 2 ) or PBS and total and phosphorylated ASK1 was measured 30 min later. D, A549 cells were transfected with a dominant negative (DN) ASK1 construct 24 h before treatment with PM 2.5 (50 g/cm 2 ) or PBS, and cell death was measured 24 h later. E, MEF from wild type and ASK1 Ϫ/Ϫ mice were treated with increasing doses of PM 2.5 , and cell death was measured 24 h later. F, wild type and ASK1 Ϫ/Ϫ MEFs were exposed to PM 2.5 (50 g/cm 2 ), and the activation of JNK was measured using a kinase assay that detects the phosphorylation of c-Jun by cell lysates. An asterisk indicates p Ͻ 0.05 when compared with untreated controls. There are a minimum of three replicates/experiment. allowed the probes to be directed to the mitochondria, cytosol, or plasma membrane while maintaining their redox-sensitive characteristics. Because the measurement is ratiometric and internally calibrated, it overcomes many of the limitations associated with other oxidant-sensitive dyes including dichlorofluorescin and dihydroethidium (34,35). We observed that PM 2.5 exposure is associated with an increase in the oxidation of this mitochondrially localized Ro-GFP. Both the ROS signal and the subsequent cell death was prevented by a superoxide dismutase/catalase mimetic, the forced overexpression of SOD1, and a knockdown of the Rieske iron-sulfur protein. These results support our conclusion that mitochondrial oxidant generation is required for PM 2.5 -induced cell death. However, our findings do not exclude the possibility that PM 2.5 also induces oxidant generation outside the cell, at the membrane, or in the cytosol. Detailed studies using spin trapping methods or employing advanced methods to detect DHE oxidation will be required to address these questions.
The potential importance of the apoptotic pathway in the development of cardiopulmonary morbidity related to PM 2.5 exposure is supported by our observation that the overexpression of SOD1 prevented the development of apoptosis in the lung as assessed using TUNEL staining and also prevented the modest increase in the permeability of the alveolar-capillary barrier observed 24 h after the instillation of P M 2.5 treatment. When administered intratracheally, adenoviral vectors primarily infect the epithelium of the airways and the alveoli (36). Inflammatory cells within the lung are resistant to adenoviral infection, and the endothelium is shielded from the virus. It is therefore reasonable to conclude that the protection we observed resulted from the overexpression of SOD1 in the alveolar epithelium.
In other models of oxidant stress, the MAPKKK ASK1 has been shown to act as a cellular redox sensor, linking intracellular oxidant generation with the apoptotic pathway (37). In the basal state, ASK1 is present in the cytosol coupled to thioredoxin (38). Thioredoxin can be oxidized at two cysteine residues, resulting in its dissociation from ASK1. Activation of ASK1 then occurs through oligomerization and auto-or crossphosphorylation (39). Once activated, ASK1 acts primarily through MKK4 and MKK7 to activate JNK or through MKK3 and MKK6 to activate p38. Activation of these kinases by ASK1 FIGURE 7. The ROS and ASK1 dependent activation of JNK is required for PM 2.5 -induced cell death. A, primary rat alveolar type II cells were treated with PM 2.5 (50 g/cm 2 ) or PBS in the presence or absence of EUK-134 (20 M), and the activation of JNK was measured 30 min later. B, primary rat alveolar type II cells were infected with adenoviruses encoding no transgene (null), SOD1, or SOD2 and 24 h later were treated with PM 2.5 (50 g/cm 2 ) or PBS. After 30 min, phosphorylated and total JNK were measured by immunoblotting. C, A549 cells were transiently transfected with a plasmid encoding a dominant negative ASK1 construct or a control plasmid (encoding GFP), and 24 h later the cells were treated with PM 2.5 (50 g/cm 2 ) or PBS and the JNK kinase activity was measured in cell lysates 2 h later. D, A549 cells infected with a retroviral vector encoding a control shRNA (D-HIF) or an shRNA against the Rieske iron-sulfur (Fe-S) protein were treated with PBS or PM 2.5 (50 g/cm 2 ), and 2 h later JNK kinase activity was measured in cell lysates. E, A549 cells were treated with SP600125 (25 M) or vehicle (DMSO) for 30 min before and during treatment with PM 2.5 (50 g/cm 2 ) or PBS, and 24 h later cell death was measured (DNA fragmentation). F, A549 cells were treated with SP600125 (25 M) or vehicle (DMSO) for 30 min before and during treatment with PM 2.5 (50 g/cm 2 ) or PBS, and 4 h later the abundance of phosphorylated (Ser-15) and total p53 was measured in cell lysates by immunoblotting. An asterisk indicates p Ͻ 0.05 when compared with untreated controls. There are a minimum of three replicates/experiment. is sustained and is responsible for apoptosis (40 -42). We observed that exposure to PM 2.5 caused the activation of ASK1 and that the inhibition of mitochondrial oxidant generation either with antioxidants or by the generation of 0 cells prevented the activation of ASK1. Genetic inhibition of ASK1 prevented PM 2.5 -induced cell death, confirming its importance in the pathway.
The JNK pathway is activated by a variety of cellular stresses including heat shock, ionizing radiation, oxidant stress, DNA damaging chemicals, reperfusion injury, mechanical shear stress, and protein synthesis inhibitors (43)(44)(45)(46). One of the mechanisms by which JNK has been shown to induce apoptosis is through activation of the tumor suppressor p53 by direct phosphorylation (47). We have previously reported that PM 2.5induced apoptosis in the alveolar epithelium requires transcriptional activation of p53 and is associated with its phosphorylation; we examined the role of JNK in the PM 2.5 -induced induction of apoptosis. We found that PM 2.5 treatment resulted in the ASK1-mediated activation of JNK and that inhibition of the JNK pathway prevented the phosphorylation of p53 at serine 18 and the subsequent cell death. Phosphorylation of p53 at serine 18 (conserved as serine 15 in the mouse) has been linked to the JNK-mediated activation of p53 in other models of oxidant stress (48,49).
In summary, we found that mitochondrial oxidant production is required for PM 2.5 -induced apoptosis in alveolar epithelial cells. The bulk of the ROS generation comes from the Q cycle in the electron transport chain, likely from the Q o site. Mitochondrially generated oxidants are required for the PM 2.5induced activation of ASK1 and cell death. The activation of ASK1 induces apoptosis by activating the JNK pathway, which may activate the intrinsic apoptotic pathway by phosphorylating p53. The importance of oxidant production in the in vivo response to PM 2.5 is supported by the observation that overexpression of SOD1 in the alveolar epithelium mitigates the PM 2.5 -induced impairment in the function of the alveolar-capillary barrier.