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We studied the role of Bim, a pro-apoptotic BCL-2 family member in Airborne particulate matter (PM 2.5 μm)-induced apoptosis in alveolar epithelial cells (AEC). PM induced AEC apoptosis by causing significant reduction of mitochondrial membrane potential and increase in caspase-9, caspase-3 and PARP-1 activation. PM upregulated pro-apoptotic protein Bim and enhanced translocation of Bim to the mitochondria. ShRNABim blocked PM-induced apoptosis by preventing activation of the mitochondrial death pathway suggesting a role of Bim in the regulation of mitochondrial pathway in AEC. Accordingly, we provide the evidence that Bim mediates PM-induced apoptosis via mitochondrial pathway.
Airborne particulate matter (PM 2.5 μm) increases morbidity and mortality from cardiopulmonary diseases resulting in an estimated 500000 deaths each year worldwide . In addition, there is evidence of an association between air pollution particles and chronic cardiopulmonary injury including lung cancer . PM is genotoxic to alveolar epithelial cell (AEC) by causing apoptosis; however, the identification of key apoptotic mediator yet remains elusive . We previously demonstrated that the mitochondria play a major role in apoptosis by releasing death-promoting molecules by activation of downstream signaling pathways [4,5]. PM exposure is a potent stimulus of mitochondrial dysfunction as detected by a change in the mitochondrial membrane potential, the release of cytochrome c and activation of caspases leading to cell death [4,6–8]. Disruption of mitochondrial electron transport can further augment reactive oxygen species (ROS) production and amplify an apoptotic stimulus due to release of mitochondrial and non-mitochondrial derived ROS [4,5,7–9].
The mitochondrial gates are crucial in initiating or restraining the downstream cascades that lead to apoptosis. The Bcl-2 family proteins consist of both pro- and anti-apoptotic members which act as gatekeepers and regulate the translocation of death-promoting molecules from the mitochondria [10,11]. The pro-apoptotic Bcl-2 family members translocate from the cytosol to the mitochondrial membrane to induce cell death [12,13]. Pro-apoptotic BCL-2 family member (Bim), in particular, initiates mitochondrial dysfunction, activates mitochondrial pathway, releases cytochrome c and activates caspases leading to apoptosis. However, the role of Bim in PM-induced apoptosis is yet unclear. In this study, we determine whether a pro-apoptotic Bim mediates PM-induced apoptosis by regulation of mitochondrial death pathway in AEC.
The PM used in our study is well characterized ambient particle 2.5 μm with known elemental analyses as described in our previous study . PM was collected by baghouse from ambient air in Dusseldorf, Germany. The particle sample was aerosolized from a turntable into a small-scale powder disperser (TSI Inc., St. Paul, MN) utilizing a high airflow to break up aggregates in the venturi throat. The outlet of the aerosol generator was attached directly to an aerodynamic particle sizer (TSI Inc.) and the aerosol was sampled on four occasions for 20 s. Data were expressed as the average mass median aerodynamic diameter from the four replicate samples. Elemental analyses of the PM were accomplished employing either infrared or thermal conductivity assays (Galbraith Labs, Knoxville, TN). Nitrogen content was measured using thermal conductivity after acid digest (Galbraith Labs). Particles contain carbon (19.70 ± 2.34%), hydrogen (1.4 ± 0.3%), nitrogen (<.05%), oxygen (14.12 ± 1.56), sulfur (2.09 ± 0.55%) and ash (63.24 ± 4.19%). Ionizable concentrations of metals associated with the particle was measured by agitation in 1.0 N HCl (1.0 mg/1.0 ml) for 1 h at room temperature, centrifuged for 1 h at 1200 × g, and the supernatant removed for analysis. Metals were individually analyzed in duplicates. Ionizable concentrations of metals include cobalt (103 ± 13 ppm), copper (48 ± 10 ppm), chromium (104 ± 23 ppm), iron (14,521 ± 572 ppm), manganese (21.3 ± 37 ppm), nickel (1519 ± 158 ppm), titanium (131 ± 45 ppm) and vanadium (2767 ± 190 ppm). All other chemicals were purchased from Sigma Chemicals.
A549 cells were obtained from the American Type Culture Collection and maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing L-glutamine (0.3 μg/ml), non-essential amino acids, penicillin (100 U/ml), streptomycin (200 μg/ml), and 10% fetal bovine serum (FBS; GIBCO) in a humidified 95% air–5% CO2 incubator at 37 °C. Rat alveolar type II epithelial cells (RLE-6TN) were obtained from the American Type Culture Collection and maintained in Ham’s F12 medium with 2 mM L-glutamine (GIBCO), bovine pituitary extract (10 μg/ml), insulin (5 μg/ml), IGF (2.5 μg/ml), transferring (1.2 μg/ml), EGF (2.5 μg/ml) and 10% fetal bovine serum in a humidified 95% air–5% CO2 incubator at 37 °C.
The coding sequence of human Bim, 5′-GACCGAGAAGGTAGACAATTG-3′, was used to generate the shRNA construct against Bim transcripts in pSilencer5.1 (Ambion Inc., Austin, TX) according to the manufacture’s instruction. The following sequence was used to create the scramble construct, 5′-CTCCGAACGTGTCACG-3′, which has no significant homology to any given gene from GEN-BANK. The retroviral supernatant derived from the above construct was prepared from Phoenix packaging cells (a kind gift from Dr. Garry Nolan at Stanford University). After infection, A549 cells were cultured in the presence of puromycin (2 μg/ml) for selection and maintaining.
AEC cell apoptosis was assessed by DNA nucleosomal fragmentation ELISA (Roche Diagnostics, Indianapolis, IN) as previously described .
Cells were treated with PM and then incubated with 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, disodium salt (MTT) (20 μl) for 3 h. An absorbance at 490 nm was measured to quantify the formazan product.
The Ψ Δm was assessed by a fluorometric assay using tetremethyl-rhodamine ethyl ester (TMRE) or Mitotracker green (Molecular Probes) followed by carbonyl cyanide trifluoromethoxyphenlhydrazone (FCCP) as previously described .
Caspase 9 and caspase 3 activity was assessed by using colorimetric activity assay kits from Upstate Laboratory as per the manufacturer’s protocol .
Cells were treated, washed and lysed. Proteins were size fractionated by 10% gel electrophoresis and transferred to nitrocellulose membranes using a semi-dry transfer (Bio-Rad). Blots were incubated with specific antibodies overnight at 4 °C and developed with an enhanced chemiluninescence detection kit (Amersham).
Cells were treated with PM and then incubated with 1 μM Mitotracker Red to stain the mitochondria. The slides were rinsed, fixed and then incubated overnight with a specific antibody (Bim: StressGen Biotech; p-Bim: Abcam Inc., MA; PARP-1: Santa Cruz Biotech) followed by DAPI for nuclear staining.
Data are reported as means ± S.E.M. Statistical analysis was done by one-way ANOVA and Tukey tests. Results were considered significant when P < 0.05.
Increasing evidence shows that proapoptotic members of the Bcl-2 family are mandatory for the initiation of apoptosis [10,14,19–21]. Bim is one of the key regulators of apoptosis [10,14]. Bim−/− lymphocytes, for example, show resistance to certain apoptotic stimuli . Induction of Bim followed by re-localization to the intracellular organelles by apoptotic stimuli is known to triggers apoptosis [19,20]. Recent studies show that Bim migration to the mitochondria is associated with apoptosis [14–19]. In particular, the accumulation and translocation of proapoptotic proteins to the mitochondria may enhance apoptosis through oxidative stress. Given the important role of Bim in the initiation of apoptosis, we determined the role of Bim in PM-induced apoptosis in A549 cells and rat alveolar type II epithelial cells.
In this study, we demonstrate that PM-induced induction of Bim and its translocation to mitochondria causes activation of mitochondrial signaling pathway leading to apoptosis. We show that downregulation of Bim by RNA interference prevents PM-induced apoptosis by inhibiting a decrease in mitochondrial membrane potential and activation of caspase-9 and caspase-3 in A549 cells. Exposure of cells to PM (100 μg/cm2) for 24 h caused a 2.5-fold increase in apoptosis as assessed by DNA Nucleosomal Fragmentation ELISA and reduction in cell viability in A549 cells (Fig. 1a and b) and rat AT II cells (Fig. 1f). We previously reported that PM at the dose of 100 μg/cm2 induces optimum level of apoptosis in A549 cells by dose response studies; therefore, this dose was used to perform all our experiments in this study . Further, we show that PM induces a significant reduction in AEC mitochondrial membrane potential and a 2–2.5-fold increase in activation of caspase-9 and caspase-3 in A549 cells, an 8-fold increase in caspase-9 and 4-fold increase in caspase-3 activation in rat AT II cells suggesting a role of mitochondrial death pathway in mediating PM-induced apoptosis in AEC (Fig. 1c–e, g–i). The generation of reactive oxygen species is one of the well-known mechanism by which PM mediates its deleterious effects in AEC [4,5,21].
The cellular machinery responsible for mitochondrial release of proapoptotic factors and mitochondrial dysfunction in oxidative stress are not well understood. Poly-(ADP-ribose)-polymerase-1 (PARP-1) is a known key regulator that modulates mitochondrial function . PARP-1 is an essential factor for DNA damage and repair. Cipriani et al. recently showed that an excessive activation of PARP rapidly triggers apoptosis by causing mitochondrial dysfunction. Impairment of the mitochondrial ATP production induces release of mitochondrial proapoptotic factors leading apoptosis . We previously reported that generation of the mitochondrial and non-mitochondrial-derived reactive oxygen species causes PM-induced apoptosis in AEC via activation of the mitochondrial death pathway [4,5]. However, the precise identification of key apoptotic mediator in PM-induced AEC apoptosis yet remains elusive. To determine the role of PARP-1 in PM-induced mitochondrial dysfunction, we examined PARP-1 activation in AEC following exposure to PM. As shown in Fig. 2a, we found that PM induces a 4-fold activation of PARP-1 in A549 cells. Upregulation of PARP-1 was associated with the induction of Bim in A549 cells exposed to PM (Fig. 2a), suggesting a possible role of PARP-1 in the release of pro-apoptotic factors in these cells .
Mitochondrial translocation of apoptosis-inducing factors is also dependent on PARP-1 . Migration of Bim to the mitochondria is an important event associated with apoptosis [18,19]. Furthermore, phosphorylation regulates the activity of these proapoptotic factors. Kurinna et al. recently demonstrated that the migration and mitochondrial translocation of Bim in response to ceramide is associated with apoptosis in A549 cells . Using immunocytochemical staining with the mitochondrial marker, mitotracker red, in this study, we examine the effects of PM in AEC. As shown in Fig. 2b and c, exposure to PM caused induction, translocation and co-localization Bim and p-Bim to the mitochondria in A549 and rat AT II cells. Further, abrogation of Bim expression by Bim shRNA inhibited PM-induced apoptosis by inhibiting the reduction of mitochondrial membrane potential and blocking activation of caspase-9 in A549 cells (Fig. 3a–c). Collectively, these data suggest that Bim plays a critical role in modulating PM-induced activation of the mitochondrial death pathway and apoptosis in AEC. A hypothetical model based on our results, showing the oxidant-dependent mechanisms by which PM modulates Bim and mitochondria-regulated apoptosis in AEC, is shown in Fig. 4.
In summary, we show that Bim mediates mitochondria-regulated particulate matter-induced apoptosis in alveolar epithelial cells. We recently showed that the mitochondria are major subcellular targets for PM as well as a source of ROS [4,5]. These previous published data coupled with our findings from this study show that impairment of mitochondrial function by particulate matter plays a critical role in PM-induced apoptosis in AEC. Strategies armed at reducing PM-induced oxidative stress may limit the toxic effects of air pollution to the lung.
Supported in part by HL010487 and ALA-Research Grant (DU).
Edited by Valdimir Skulachev