Silica-Induced Caspase Activation in Mouse Alveolar Macrophages Is Dependent upon Mitochondrial Integrity and Aspartic Proteolysis

M. Thibodeau*, C. Giardina{dagger} and A. K. Hubbard*,1

* Departments of Pharmaceutical Sciences and {dagger} Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269

Received May 22, 2003; accepted June 18, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although silica has been documented to cause apoptotic cell death, the cellular pathways leading to caspase activation have not been extensively investigated. Here we demonstrate in a mouse macrophage cell line (MH-S cells) that {alpha}-quartz silica exposure (12.5 µg/cm2 to 50 µg/cm2) elicited activation of both caspase 3 and caspase 9, whereas anatase titanium dioxide (TiO2), a non-fibrogenic particle, did not. Silica exposure in vitro also induced apoptosis after 6 h, as measured by the appearance of subdiploid cell fragments in a flow cytometric analysis. Exposure to TiO 2 did not elicit significant apoptosis. Silica-induced apoptosis and caspase 3 activation were, in part, caspase 9 dependent, as determined by their sensitivity to either a general caspase inhibitor (Z-VAD-FMK) or a specific caspase 9 inhibitor (Z-LEHD-FMK). Silica exposure in vitro also elicited significant mitochondrial depolarization after 2 and 6 h of exposure. Cyclosporin A, an inhibitor of the mitochondrial permeability pore, partially decreased mitochondrial depolarization, caspase 3 activation, and caspase 9 activation, suggesting a role for mitochondrial dysfunction in these events. Pepstatin A, an inhibitor of cathepsin D, also decreased mitochondrial depolarization, caspase 3 activation, and caspase 9 activation, whereas leupeptin, an inhibitor of cathepsin B, had no effect. These data suggest that short-term silica exposure in vitro induces both caspase 3 and caspase 9 activity, which appears to participate in apoptosis. Activation of these caspases seems to be dependent, in part, on aspartic proteolysis and loss of mitochondrial integrity.

Key Words: silica; apoptosis; caspase 3; caspase 9; mitochondria; cathepsins.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhalation of crystalline silica in the workplace is associated with silica-induced lung injury and the development of silicosis (ATS Committee, 1997Go). Although silica has been documented to cause necrotic cell death (Carter and Driscoll, 2001Go: Porter et al., 2002Go), evidence exists that silica can also initiate caspase activation and apoptosis. However, the identity of caspases activated, their relationship to apoptosis, and cellular pathways leading to caspase activation have not been extensively investigated. It is important to determine the role and pathways of caspase activation and apoptosis in silica-induced lung injury, since these events may be targets for therapeutic intervention.

Caspases exist as relatively inactive precursors or procaspases that are converted into their active forms by proteolytic cleavage at internal aspartic acid residues. All caspases show a high degree of specificity, with an absolute requirement for cleavage after an aspartic acid residue and a recognition sequence of at least four amino acids N-terminal to the cleavage site. This specificity is not only important in the cleavage of pro-caspases to their active form, but has been exploited in the design of highly specific and effective enzyme inhibitors (Grutter, 2000Go).

The mammalian caspase family contains 14 members, a subset of which participates in apoptosis, with the remainder involved in the processing of proinflammatory cytokines (Chang and Yang, 2000Go). Death-inducing caspases interact in a coordinated cascade to propagate cell signaling pathways leading to eventual apoptosis. Effector caspases 3, 6, and 7 are responsible for cleaving structural elements, nuclear proteins, and signaling proteins (Krammer, 1999Go). Nuclear condensation is apparent with fragmentation of the nucleus and DNA and formation of apoptotic bodies. Caspase 8, coupled extrinsically to death receptors, and caspase 9, regulated intrinsically by mitochondrial dysfunction, activate caspase 3 to initiate apoptotic events.

The participation of caspase activation in silica-induced apoptosis and inflammation has also been investigated both in vitro and in vivo. Exposure in vitro of mouse macrophages (Sarih et al., 1993Go) or human alveolar macrophages (AMs) (Iyer et al., 1996Go) has been reported to elicit apoptosis (Sarih et al., 1993Go). In human AMs treated with a nonspecific (pan) caspase inhibitor, carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK), there was a decrease in silica-induced apoptosis (Iyer et al., 1996Go). These authors later confirmed the involvement of caspase 3 in silica-induced apoptosis in human AM using a specific inhibitor of caspase 3, carbobenzoxy-asp-glu-val-asp-[O-methyl]-fluoromethylketone (Z-DEVD-FMK) (Iyer and Holian, 1997Go). Silica also stimulated activation of caspases 1, 3, and 6 in a mouse alveolar macrophage cell line, MH-S (Chao et al., 2001Go) and caspases 3 and 9-like activity in rat AMs (Shen et al., 2001Go).

The intrinsic pathway of apoptosis is usually driven by alterations in inner and/or outer mitochondrial membrane permeability, which can be measured by release of intermembranous proteins (i.e., cytochrome c) or depolarization of the inner mitochondrial membrane transmembrane potential (Castedo et al., 2000Go). Frequently preceding the release of cytochome c is mitochondrial membrane permeabilization (MMP), also known as the mitochondrial permeability transition (MPT) (Ferri and Kroemer, 2001Go). Changes in MMP can lead to the activation of caspase 9 and the subsequent activation of caspase 3 (Li et al., 1997Go; Srinivasula et al., 1998Go). Mechanisms that may contribute to MMP vary with apoptotic stimulus, but may include alterations in Bcl-2 proteins, reactive oxygen species, calcium, ceramide metabolites, and more recently, endolysosomal cathepsins. Lysosomal damage can induce cytochrome c release from mitochondria leading to the activation of both caspases 3 and 9 (Reiners et al., 2002Go). The lysosomal cysteine protease, cathepsin B, and aspartic protease, cathepsin D, have both been implicated in the induction of apoptosis. In response to oxidative stress, cathepsin D translocation from lysosomes to the cytosol can precede cytochrome c release and loss in MMP (Roberg et al., 1999Go). In this model, pepstatin A, a specific inhibitor of cathepsin D, prevented the mitochondrial dysfunction induced by oxidative stress (Roberg et al., 1999Go). Similarly, lysosomal cathepsin B can induce the mitochondrial pathway (Guicciardi et al., 2000Go). Silica can also elicit lysosomal injury and increase cathepsin D activity (Jajte et al., 1988Go; Sjostrand and Rylander, 1984Go), which may also contribute to caspase activation.

Despite the significant work accomplished recently in dissecting the relationship between silica-induced caspase activation and cell injury, significant gaps in knowledge remain. It is not clear which caspases are activated or the cellular pathways leading to their activation. Here we address these questions to determine the role and regulation of caspase activation in response to silica.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell line.
The mouse alveolar macrophage cell line MH-S (ATCC CRL-2019) was cultured at 37°C with 5% CO2 in RPMI 1640 media supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 55 µM 2-mercaptoethanol, 10% fetal bovine serum, 50 units/ml penicillin G, and 50 µg/ml streptomycin (Gibco BRL Life Technologies, Grand Island, NY).

Exposures in vitro to silica.
Prior to treatment with particle, cells were plated in 6 or 24 well plates at approximately 2 x 105 cells/cm2 in culture media and allowed to adhere for 2 h, after which culture media was replaced. The next day, cells were stimulated in RPMI 1640 media only with or without {alpha}-quartz silica (Min-U-Sil 5; Pennsylvania Glass and Sand Corp.; Pittsburgh, PA) or anatase titanium dioxide (TiO2; Sigma Chemical Co.; St. Louis, MO). For inhibition of caspases, cells were pretreated for 1 h with 50 µM Z-VAD-FMK (Enzyme Systems, Inc., Livermore, CA), 50 µM carbobenzoxy-leu-glu-[O-methyl]-his-asp-[O-methyl]-fluoromethylketone (Z-LEHD-FMK) (Enzyme Systems, Inc., Livermore, CA) or DMSO (0.25%) and then concomitantly with silica. For inhibition of mitochondrial permeability transition complex, cells were pretreated for 1 h with 10 µM cyclosporin A (Sigma, St. Louis, MO) or DMSO (0.25%) and then concomitantly with silica. For inhibition of cathepsin B and D, respectively, cells were pretreated for 16 h with 0.1 to 100 µM pepstatin A or leupeptin (Sigma, St. Louis, MO) or DMSO (0.25%) and then concomitantly with silica.

Percent cytotoxicity (lactate dehydrogenase [LDH] release).
Cells were treated with {alpha}-quartz and evaluated for cell death using the Cytotox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI) as described by the manufacturer. For inhibition of caspases, cells were pretreated for 1 h with 50 µM Z-VAD-FMK (Enzyme Systems, Inc., Livermore, CA) or DMSO (0.25%) and then concomitantly with silica. Following treatment, cell supernatants and intact cells were separated by centrifugation at 120 x g for 6 min at 4°C. Cell supernatants containing released LDH were saved. Cell pellets (intact cells) were incubated with the provided lysis solution for 45 min at 37°C, followed by extraction of the cell lysates by centrifugation at 120 x g for 6 min at 4°C. Cell supernatants and lysates diluted 1:5 in phosphate-buffered saline (PBS) were incubated with the provided LDH substrate for 30 min at room temperature, followed by addition of the provided stop solution. LDH activity (IU/ml) was calculated after measurement of the OD490nm. Percentage cytotoxicity was calculated as the LDH IU/ml supernatant/(LDH IU/ml supernatant + LDH IU/ml intact cells).

Flow cytometric analysis for caspase activity in vitro.
Following treatment with {alpha}-quartz, the media was replaced with RPMI-1640 containing a carboxyfluorescein conjugated substrate for caspase-3-like activity (CaspaTag, 7.5 µM FAM-DEVD-FMK; Intergen Co., Purchase, NY). After 1 h incubation at 37°C, nonadherent (floaters) and adherent cells were harvested for flow cytometric analysis. Adherent cells were gently detached from tissue culture plates (using PBS with 0.5 mM EDTA), combined with nonadherent cells, washed, centrifuged at 150 x g 4°C for 5 min, resuspended in wash buffer, and fixed with paraformaldhyde. Cells staining for caspase activity were detected using a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA). Cells were gated to exclude silica particles and small sized cell debris (low forward scatter). Cells with increased caspase activity were expressed as a percentage of total gated cells.

Western immunoblot analysis for caspase cleavage in vitro.
After stimulation with either {alpha}-quartz or TiO2, adherent and floating cells were collected and cell extracts were prepared in PBS pH 7.6 with 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS containing freshly added protease inhibitors: 2 mM phenylmethanesulfonyl fluoride (PMSF), 10 µg/ml apoprotinin, 10 µg/ml pepstatin, and 10 µg/ml leupeptin hemisulfate (Sigma, St. Louis, MO). Extracts were incubated on ice (1 h), centrifuged at 10,000 x g for 10 min 4°C, and the supernatants electrophoresed immediately or stored at -80°C. Samples were resolved on a 12.5% or a 15% SDS–polyacrylamide gel and electrotransfered to nitrocellulose. Blots were blocked in TBS/0.1% Tween 20/5% (w/v) low fat milk for 1 h at room temperature (RT). Immunodetection of specific caspases was conducted with rabbit anti-caspase 3 (Cell Signaling Technology; Beverly, MA), rabbit anti-caspase 9 (Cell Signaling Technology; Beverly, MA), or rabbit anti-caspase 8 (Santa Cruz Biotechnology, Inc; Santa Cruz, CA; NeoMarkers Inc., Freemont, CA) and a peroxidase-conjugated goat anti-rabbit secondary antibody (Santa Cruz Biotechnology, Inc; Santa Cruz, CA). Normalization of samples was performed by loading similar amounts of protein (extracts equivalent to 1 to 2 x 104 cells/µl) and by reprobing the blots with goat anti-actin antibody (Santa Cruz Biotechnology, Inc; Santa Cruz, CA) followed by peroxidase-conjugated donkey anti-goat antibody (Santa Cruz Biotechnology, Inc; Santa Cruz, CA). Detection of immunopositive bands was performed using luminol reagents (Santa Cruz Biotechnology, Inc; Santa Cruz, CA) and a Kodak Image Station 440CF.

Measure of apoptosis in vitro.
After stimulation with either silica or TiO2 particles, cells were analyzed for DNA fragmentation into oligonucleosomes by flow cytometry using cell cycle analysis for subdiploid DNA content (Lecoeur, 2002). Adherent cells were gently detached from tissue culture plates (using PBS with 0.5 mM EDTA), combined with nonadherent cells, centrifuged 6 min at 150 x g 4°C, washed, and again centrifuged. Cells were resuspended, fixed, and permeabilized overnight in 70% ethanol at 4°C. Cells were again centrifuged, resuspended in PBS with 0.1% Triton X-100 containing 200 µg/ml RNAase A (Sigma Chemical Co., St. Louis, MO) and 20 µg/ml propidium iodide (Sigma Chemical Co., St. Louis, MO), and incubated 30 min 37°C in the dark. Cell cycle analysis was performed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). Cells were gated to exclude silica particles and cellular debris having small size (low forward scatter), and the subdiploid cells expressed as a percentage of total gated cells.

Determination of mitochondrial permeabilization.
Following treatment with {alpha}-quartz, the cells were evaluated by flow cytometry for mitochondrial staining with the lipophilic, cationic dye tetremethylrhodamine ethyl ester (TMRE). The adherent cells were gently detached from tissue culture plates (PBS with 0.5 mM EDTA), combined with nonadherent cells, centrifuged 6 min at 150 x g 4°C, and resuspended in PBS. Cells were stained with 200 nM TMRE and incubated 15 min 37°C in the dark. Positive control cells were stained in the presence of carbonyl cyanide trifluoromethoxyphenylhydrazone (10 µM FCCP; Sigma, St. Louis, MO), a protonophore causing mitochondrial depolarization in approximately 70% of the cells (data not shown). After incubation, cells were immediately placed on ice and evaluated for fluorescence using a using a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA). Cells were gated to exclude silica particles and small-sized cell debris (low forward scatter). Cells with decreased FL2 fluorescence (mitochondrial depolarization) were expressed as a percentage of total gated cells.

Statistical analysis.
Data are expressed as mean ± standard error. Differences between groups were evaluated by analysis of variance using the Student-Newman-Keuls procedure to correct for multiple comparisons (GraphPad Prism, v. 3.03). A p value of less than 0.05 was considered significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Silica Induces Caspase-Dependent Cell Death
MH-S mouse macrophages were exposed for 6 h to {alpha}-quartz at either 12.5 µg/cm2 or 50 µg/cm2 and evaluated for cell membrane integrity and viability by LDH release (Table 1Go). This measure is the result of both cell necrosis and late stage apoptosis (Gomez-Lechon et al., 2002Go). Only incubation with the higher dose of silica (50 µg/cm2) elicited LDH release above that seen in media-stimulated cells. This result indicates that silica induces necrosis, or apoptosis with secondary necrosis, in ~40% of cells. Addition of the pan caspase inhibitor, Z-VAD-FMK, to cells incubated with 50 µg/cm2 silica significantly reduced LDH release, indicating a population of cells undergoing caspase-dependent cell death (apoptosis) at 6 h.


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TABLE 1 Silica-Induced Cell Death
 
Silica-Induced Apoptosis in Vitro Is Caspase Dependent
To confirm that silica elicits apoptotic cell death, MH-S mouse macrophages were incubated with two concentrations of {alpha}-quartz silica (12.5 or 50 µg/cm2) for 6, 12, or 24 h and assessed for DNA fragmentation, a marker of apoptosis evidenced by the percentage of cells in the subdiploid fraction of the cell cycle (Fig. 1AGo). Silica induced apoptosis in an increasing number of cells with increased dose and with increased time of exposure. To confirm the specificity of these effects, cells were incubated with either silica or anatase titanium dioxide (50 µg/cm2) for 6 h. After incubation with silica, a significant percentage of cells (~13%) demonstrated an increase in this subdiploid population, whereas incubation with TiO2 elicited only a minor increase (~3%) (Fig. 1BGo). Silica-induced apoptosis appeared to be dependent upon caspase activation, as preincubation with the pan caspase inhibitor, Z-VAD-FMK, significantly reduced the percentage of apoptotic cells (Figs. 2AGo and 2BGo).



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FIG. 1. Silica exposure in vitro induces apoptosis. (A)MH-S cells were exposed in vitro to {alpha}-quartz silica (12.5 µg/cm2 or 50 µg/cm2) or media for 6, 12, or 24 h. After stimulation, the cells were processed for DNA content by cell cycle analysis. Apoptotic (subdiploid) cells are expressed as a percentage of total gated cells and as the mean ± SE (n = 3 wells). *Significantly different from media exposed cells; p < 0.05. #Significantly different from cells exposed to silica at 12.5 µg/cm2; p < 0.05. (B) MH-S cells were exposed in vitro to either {alpha}-quartz silica or TiO2 (50 µg/cm2) for 6 h. After stimulation, the cells were processed for DNA content by cell cycle analysis. Apoptotic (subdiploid) cells are expressed as a percentage of total gated cells and as the mean ± SE (n = 3 wells). *Significantly different from media exposed cells; p < 0.05. #Significantly different from cells exposed to silica; p < 0.05.

 


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FIG. 2. Silica-induced apoptosis is caspase dependent. MH-S cells were exposed in vitro to 50µg/cm2 {alpha}-quartz with or without 50 µM Z-VAD-FMK for 6 h. After stimulation, the cells were processed for DNA content by cell cycle analysis. (A) Representative histograms showing the subdiploid cells (M1 fraction). (B) Apoptotic (subdiploid) cells are expressed as a percentage of total gated cells and as the mean ± the standard error (n = 3 wells). *Significantly different from media exposed cells; p < 0.05. #Significantly different from silica-exposed cells in the absence of Z-VAD-FMK; p < 0.05.

 
Silica Induces Caspase 3 and 9 Activation in Vitro
To examine caspase activation in MH-S mouse macrophages in greater detail, cells were exposed to {alpha}-quartz (50 µg/cm2) for 6 h and then incubated with the fluoresceinated caspase 3 substrate, FAM-DEVD-FMK. Flow cytometric analysis revealed a significant increase in the percentage of gated cells (~14%) that cleaved this caspase 3 substrate (Fig. 3AGo), indicating caspase-3-like activity. Activation of caspase 3 by silica-exposed cells (50 µg/cm2) was confirmed by Western immunoblot, demonstrating the generation of an antibody-reactive cleavage product at 17 kD from the precursor enzyme of 32 kD (Fig. 3BGo). This cleavage product was first apparent 2 h after silica exposure and remained prominent through 8 h. Cells from media-stimulated cultures did not evidence increased caspase 3-like activity (Fig. 3AGo) or the 17 kD cleavage products (Fig. 3BGo). Incubation of MH-S cells with increasing concentrations of silica demonstrated cleavage of caspase 3 at 12.5 µg/cm2, whereas exposure to TiO2, a non fibrogenic particle, at similar concentrations did not appear to activate caspase 3 (Fig. 3CGo).



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FIG. 3. Silica exposure in vitro activates caspase 3. (A) Flow cytometric analysis for caspase-3-like activity (FAM-DEVD-FMK cleavage) by MH-S cells exposed in vitro to 50 µg/cm2 {alpha}-quartz for 6 h. Cells with caspase-3-like activity are expressed as a percentage of total gated cells and as the mean ± the standard error (n = 3 wells). *Significantly different from media exposed cells (p < 0.05). (B) Western blot analysis for active caspase 3 (p17) in cell lysates from MH-S cells exposed in vitro to 50 µg/cm2 {alpha}-quartz or media for 0 to 8 h. Detection of actin was used to ensure equal sample loading per lane. Representative of three separate experiments. (C) Western blot analysis for active caspase 3 (p17) in cell lysates from MH-S cells exposed for 6 h to {alpha}-quartz or TiO2 (6.25 µg/cm2 to 50 µg/cm2). Detection of actin was used to ensure equal sample loading per lane. Representative of two separate experiments.

 
Exposure of MH-S mouse macrophages to silica also elicited activation of caspase 9, as evidenced by generation of cleavage products at both 37 and 39 kD from the 49 kD procaspase after 2 to 4 h (Fig. 4AGo). The p39 cleavage product was first evident at 2 h, and the p37 cleavage product increased in intensity at 4 h. Exposure of cells to TiO2 did not elicit processing of caspase 9, as evidenced by the lack of induction of either the p39 or p37 cleavage products (Fig. 4BGo).



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FIG. 4. Silica exposure in vitro activates caspase 9. (A) Western blot analysis for active caspase 9 (p37, p39; arrows) in cell lysates from MH-S cells exposed in vitro to 50 µg/cm2 {alpha}-quartz or media for 0 to 8 h. Detection of actin was used to ensure equal sample loading per lane. Representative of two separate experiments. (B) Western blot analysis for active caspase 9 (p37, p39; arrows) in cell lysates from MH-S cells exposed for 6 h to {alpha}-quartz or TiO2 (6.25 µg/cm2 to 50 µg/cm2). Detection of actin was used to ensure equal sample loading per lane. Representative of two separate experiments.

 
Silica-Induced Apoptosis and Caspase 3 Activation Are Caspase 9 Dependent
The generation of caspase 3 and resulting apoptosis was also dependent upon caspase 9 activity. Figure 5Go demonstrates a reduction in caspase 9 (Fig. 5AGo) and caspase 3 (Fig. 5BGo) activation following incubation with the caspase 9 specific inhibitor, Z-LEHD-FMK, as well as with the pan caspase inhibitor, Z-VAD-FMK. This reduction in caspase activation by these inhibitors also led to a significant decrease in apoptosis (Fig. 5CGo).



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FIG. 5. Silica-induced caspase activation and apoptosis is dependent upon caspase 9 activity. MH-S cells were exposed in vitro to 50 µg/cm2 {alpha}-quartz with or without 50 µM Z-VAD-FMK or 50 µM LEHD-FMK for 6 h. Cell lysates were examined by Western blot analysis for antibody reactive caspase 9 cleavage products (p39, p37; arrows). Detection of actin was used to ensure equal sample loading per lane. Representative of two separate experiments. MH-S cells were exposed in vitro to 50 µg/cm2 {alpha}-quartz with or without 50 µM Z-VAD-FMK or 50 µM Z-LEHD-FMK for 6 h. Cell lysates were examined by Western blot analysis for antibody reactive caspase 3 cleavage products (p17). Detection of actin was used to ensure equal sample loading per lane. Representative of two separate experiments. MH-S cells were exposed in vitro to 50 µg/cm2 {alpha}-quartz with or without 50 µM Z-VAD-FMK or 50 µM LEHD-FMK for 6 h. After stimulation the cells were processed for DNA content by cell cycle analysis. Apoptotic (subdiploid) cells are expressed as a percentage of total gated cells and as the mean ± the standard error (n = 3 wells). *Significantly different from media exposed cells; p < 0.05. #Significantly different from cells exposed to silica; p < 0.05.

 
Silica exposure of MH-S cells did not elicit the cleavage of caspase 8 over a period of 0.5 to 8 h at any concentration of silica (6.25 µg/cm2 to 50 µg/cm2) as evaluated by immunoblotting (data not shown).

Silica Induces Mitochondral Permeability Transition
Activation of caspase 9 is usually reflective of perturbation of the intrinsic mitochondrial pathway of apoptotic cell death. Figure 6Go demonstrates that exposure of MH-S cells at 50 µg/cm2 for either 2 (Figs. 6AGo and 6CGo) or 6 h (Figs. 6BGo and 6DGo) significantly increased the percentage of cells with depolarization of the inner mitochondrial transmembrane potential ({Delta}{Psi}m). This change in MMP induced by silica exposure could be partially prevented by 10 µM cyclosporin A (Fig. 7AGo), a known inhibitor of the MPT complex. Cyclosporin A (10 µM) also significantly reduced the cleavage maturation of both caspase 9 (Fig. 7BGo), and caspase 3 (Fig. 7CGo) indicating a role for the MPT complex in the activation of these caspases.



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FIG. 6. Silica exposure in vitro induces mitochondrial permeability transition. Cells were incubated with silica (50 µg/cm2 {alpha}-quartz) for 2 h (A) or 6 h (B) and examined by flow cytometry for depolarized mitochondria ({Delta}{Psi}m). Data are representative histograms of the depolarized fraction (M2). Data are also expressed as a percentage of total gated cells with decreased FL2 fluorescence (mitochondrial depolarization) following 2 h silica exposure (C) or 6 h exposure (D) as the mean ± the standard error (n = 3 wells). *Significantly different from media exposed cells; p < 0.05.

 


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FIG. 7. Silica-induced caspase activation is dependent upon mitochondrial permeability transition. (A) MH-S cells were exposed in vitro to 50 µg/cm2 {alpha}-quartz (6 h) with or without 10 µM cyclosporin A. After stimulation the cells were processed for mitochondrial depolarization ({Delta}{Psi}m) by flow cytometry. Data are expressed as a percentage of total gated cells with decreased FL2 fluorescence (mitochondrial depolarization) as the mean ± SE (n = 3 wells); *Significantly different from media exposed cells; p < 0.05. #Significantly different from silica-exposed cells in the absence of cyclosporin A; p < 0.05. (B) MH-S cells were exposed in vitro to 50 µg/cm2 {alpha}-quartz (6 h) with or without 10 µM cyclosporin A. Cell lysates were examined by Western blot analysis for antibody reactive caspase 9 (p39, p37; arrows) cleavage products. Detection of actin was used to ensure equal sample loading per lane. Representative of two separate experiments. (C) MH-S cells were exposed in vitro to 50 µg/cm2 {alpha}-quartz (6 h) with or without 10 µM cyclosporin A. Cell lysates were examined by Western blot analysis for antibody reactive caspase 3 cleavage products (p17). Detection of actin was used to ensure equal sample loading per lane. Representative of two separate experiments.

 
Endolysosomal Proteases Participate in Silica-Induced Mitochondrial Permeability Transition and Caspase Activation
Silica is known to disrupt the endolysosomal compartment of cells (Jajte et al., 1988Go; Sjostrand and Rylander, 1984Go), which in some models of apoptosis is known to contribute to mitochondrial dysfunction. To explore if endolysosomal cathepsin proteases contribute to silica-initiated MPT and the activation of caspases, cells were preincubated with either pepstatin A (0.1 to 100 µM) or leupeptin (0.1 to 100 µM). Pretreatment of cells with the aspartic protease (cathepsin D) inhibitor pepstatin A prior to silica exposure (50 µg/cm2) not only partially inhibited mitochondrial membrane depolarization (Fig. 8AGo), but also reduced activation of both caspase 3 and caspase 9 (Figs. 8BGo and 8CGo). These changes were specific to pepstatin A, as leupeptin, an inhibitor of cathepsin B, did not elicit these effects (Figs. 8BGo and 8CGo).



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FIG. 8. Silica-induced caspase activation is dependent upon cathepsin D activity. (A) MH-S cells were exposed in vitro to 50 µg/cm2 {alpha}-quartz (6 h) with or without 1 µM pepstatin A. After stimulation the cells were processed for mitochondrial depolarization ({Delta}{Psi}m) by flow cytometry. Data are expressed as a percentage of total gated cells with decreased FL2 fluorescence (mitochondrial depolarization) as the mean ± the standard error (n = 3 wells); *Significantly different from media exposed cells; p < 0.05. #Significantly different from silica-exposed cells in the absence of pepstatin A, p < 0.05. MH-S cells were exposed in vitro to 50 µg/cm2 {alpha}-quartz (6 h) with or without pepstatin A or leupeptin. Cell lysates were examined by Western blot analysis for antibody reactive caspase 9 (p39, p37; arrows) cleavage products. Detection of actin was used to ensure equal sample loading per lane. Representative of 2 separate experiments. (C) MH-S cells were exposed in vitro to 50 µg/cm2 {alpha}-quartz (6 h) with or without pepstatin A or leupeptin. Cell lysates were examined by Western blot analysis for antibody reactive caspase 3 (p17) cleavage products. Detection of actin was used to ensure equal sample loading per lane. Representative of two separate experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure of cells to {alpha}-quartz silica has been documented to cause cell death, often measured by LDH release (Table 1Go) (Carter and Driscoll, 2001Go; Porter et al., 2002Go). However, over the past several years, exposure to silica has also been associated with caspase activation and apoptosis. We have demonstrated apoptosis in MH-S mouse macrophages following exposure to silica particles (Fig. 1AGo), but not to TiO2 (Fig. 1BGo). This apoptotic response appeared to be dependent upon caspase activation, since incubation of cells with a pan caspase inhibitor (Z-VAD-FMK) significantly reduced apoptosis (Fig. 2Go). Activation of caspase 3 and caspase 9 was found to be elicited by silica exposure in vitro (Figs. 3Go and 4Go). Moreover, detection of caspase 3 and caspase 9 cleavage products (Fig. 3Go) appeared to be sensitive markers of early changes and at silica concentrations (12.5 µg/cm2) which did not elicit cell membrane permeability (necrotic cell death) (Table 1Go). Our work is the first to demonstrate that processing of caspase 9 and caspase 3 and resulting apoptosis are specific events noted after exposure to fibrogenic silica particles but not to TiO2 (Figs. 3CGo and 4BGo), a nonfibrogenic particle. Our work also demonstrates the dependence of apoptosis on caspase 9 activation, since a selective caspase 9 inhibitor (Z-LEHD-FMK) significantly reduced silica-induced apoptosis. The caspase 9 inhibitor also suppressed caspase 3 activation, implicating caspase 9 as an upstream caspase in silica-induced apoptosis (Fig. 5Go).

Our work also demonstrates that cleavage activation of caspase 9 in MH-S mouse macrophages results in the detection of both p37 and p39 products. Caspase 9 can be alternatively processed to either a p37 subunit or p39 subunit, depending upon whether cleavage occurs at Asp 353 (p37) or Asp 368 (p39) residues. In mice, procaspase 9 (49 kD) is cleaved by the apoptosome at Asp 353 to form p37 (Fujita et al., 1999Go, 2000Go; Little and Mirkes, 2002Go; Srinivasula et al., 1998Go), which is usually regulated by the release of mitochondrial intermembranous cytochrome c. Active caspase 9 can then cleave the effector caspase 3 (32 kD) into active p20 and p12 subunits (Bratton et al., 2001Go). Amplifying the processing of caspase 9 is a feedback loop involving active caspase 3. In mice, caspase 3 can cleave procaspase 9 at the Asp 368 residue forming the active p39 product (Little and Mirkes, 2002Go). In our model, silica appears to activate the autocatalytic cleavage of procaspase 9 as well as its cleavage by caspase 3. Production of the p39 cleavage product is specific to silica-treated cells, since in media- or TiO2-treated cells there is no expression of p39.

Our work not only provides new information on silica-induced caspase activation and apoptosis, but also extends previous reports. Chao et al.(2001)Go detected silica-induced apoptosis in MH-S cells by the cell death ELISA and caspase 1, 3, and 6 activation by cleavage of chromogenic substrates and by Western immunoblot (caspase 3 only). Inducing apoptosis in approximately 15% of macrophages exposed to silica at 50 µg/cm2 for 6 h (Fig. 1Go) is comparable to reports in rat AMs of ~8% apoptotic cells after silica exposure at 30 µg/cm2 and ~ 26% apoptotic cells after silica exposure at 100 µg/cm2 silica for 9 h (Wang et al., 2002Go). Our work also extends previous studies in which neither TiO2 (Iyer et al., 1996Go; Zhang et al., 2002Go) nor amorphous silica (Iyer et al., 1996Go) elicited apoptotic cell death. We find that TiO2 exposure does not activate either caspase 3 or caspase 9. When human AMs were treated with a pan caspase inhibitor (Z-VAD-FMK), a decrease in both silica-induced apoptosis and IL-1b release was observed (Iyer et al., 1996Go). These authors later confirmed the involvement of caspase 3 in silica-induced apoptosis in human AM using a caspase 3 specific inhibitor (Z-DEVD-FMK) (Iyer and Holian, 1997Go). Shen et al.(2001)Go also noted in rat AMs a role for caspase 3 in silica-induced apoptosis through the use of a caspase 3 inhibitor.

Our work is also the first to demonstrate that processing of caspase 3 and caspase 9 following silica exposure is, in part, due to changes in inner mitochondrial membrane transmembrane potential. The intermembranous mitochondrial space can release cytochrome c, which translocates to the cytosol and complexes with apoptosis protease activating factor-1 (APAF-1) and dATP/ATP (Srinivasula, 1998Go; Zou, 1999Go). Frequently preceding the release of cytochome c is MPT (Ferri and Kroemer, 2001Go). MPT is a change in permeability across the inner mitochondrial membrane that allows solutes less than 1.5 kD to pass, leading to depolarization of mitochondria (Bernardi, 1999Go; Lemasters et al., 2002Go). Our results with TMRE staining of silica-exposed cells indicate induction of mitochondrial depolarization concurrent with the activation of caspase 3 and 9. Current theory presumes the MPT to be a multiprotein permeability transition core complex (PTCC) created at Hackenbrock’s contact sites between the inner and outer mitochondrial membranes (Ferri and Kroemer, 2001Go; Lemasters et al., 2002Go). The PTCC often includes the matrix protein cyclophilin D, making these pores susceptible to inhibition by cyclosporin A. (Ferri and Kroemer, 2001Go). MPT can, therefore, be regulated by cyclosporin A (CsA) or independent from the effects of CsA (He and Lemasters, 2002Go). In our study, the majority (70%) of depolarized cells exposed to silica are not regulated, whereas a subpopulation (approximately 30%) has regulated mitochondrial depolarization (Fig. 7Go). Our work clearly demonstrates that silica exposure initiates MPT (Fig. 6Go) that leads to caspase activation (Fig. 7Go). In addition, partial prevention of mitochondrial depolarization with either cyclosporin A or pepstatin A reduced both caspase 9 and caspase 3 activation (Figs. 8BGo and 8CGo). These data would suggest that preventing mitochondrial depolarization would alter silica-induced apoptosis. These results demonstrate the inter relationship between aspartic proteases (e.g., lysosomal cathepsin D), mitochondrial dysfunction, and caspase activation in lung alveolar macrophages exposed to silica particles (Fig. 9Go). Other groups have suggested a role for death receptors in the initiation of silica-induced apoptosis (Borges et al., 2001Go, 2002Go). However, the extrinsic pathway of apoptotic cell death probably does not contribute significantly in our model, because we could not detect cleavage activation of caspase 8 following silica exposure at any time points preceding activation of caspase 3 (data not shown).



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FIG. 9. Proposed pathway for silica-induced caspase activation and apoptosis in alveolar macrophages. Silica particles activate cathepsin D (lysosomes) or cathepsin E (plasma membrane, endosomes, or endoplasmic reticulum), contributing to mitochondrial permeability transition. The mitochondrial permeability transition complex initiates the intrinsic pathway to activate caspase 9 and the subsequent processing of caspase 3 leading to apoptosis.

 
The MPT triggered by silica may also result from the activation of lysosomal cathepsins (Ferri and Kroemer, 2001Go; Reiners et al., 2002Go). Previous studies using either pepstatin A, a specific inhibitor of aspartic proteases (among which is cathepsin D), or leupeptin, a broad inhibitor of serine and cysteine proteinases (among which is cathepsin B), have demonstrated a role for both cathepsin D and cathepsin B in mitochondrial-mediated apoptosis induced by various stimuli. [Cathepsins are not believed to directly activate or cleave procaspases (Stoka et al., 2001Go).] In response to oxidative stress, cathepsin D translocation from the lysosome to the cytosol can precede cytochrome c release and loss in mitochondrial membrane potential (Roberg et al., 1999Go). In silica-exposed alveolar macrophages, we show that pepstatin A, but not leupeptin, potently inhibits activation of both caspase 3 and caspase 9 (Figs. 8BGo and 8CGo), and also inhibits the MTP with the same efficiency as cyclosporin A (Fig. 8AGo). Since other members of the aspartic protease family are secretory proteins (Cooper, 2002Go), these findings suggest a released endolysosomal cathepsin, such as cathepsin D, contributes to the initiation of caspase activation through mitochondria dysfunction. Although the mechanism by which cathepsins produce mitochondrial dysfunction is uncertain, they have been shown to cleave the Bcl-2-related Bid protein to a proapoptotic form, promoting mitochondrial depolarization and cytochrome c release (Roberg et al., 1999Go).

In addition to a role for cathepsin D in mitochondrial dysfunction, reactive oxygen and nitrogen species are well recognized to induce the mitochondrial pathway of apoptosis (Lemasters et al., 2002Go), and future studies will address this in our model. Shen et al.(2001)Go noted in rat AMs, a temporal pattern of events over the first 4 h of silica exposure beginning with reactive oxygen species (ROS) formation, caspase 9 and caspase 3 activation, PARP cleavage, and DNA fragmentation. Studies with other toxic particles also suggest a mitochondrial-mediated pathway of apoptosis. In the human alveolar epithelial cell line A549, asbestos elicited mitochondrial dysfunction, translocation of cytochrome c, and the activation of caspase-9-like activity, changes that did not occur with non-fibrogenic TiO2 or glass beads (Kamp et al., 2002Go).

Although silica has been documented to elicit marked pulmonary inflammation and cell death, it appears that silica can also induce apoptotic cell death through changes in mitochondrial membrane integrity and caspase activation driven, in part, by lysosomal cathepsin activity. The biologic significance of silica-induced caspase activation and resulting apoptosis may be in the resolution of these inflammatory lesions through the elimination of damaged or injured cells (Shen et al., 2001Go) or in a proinflammatory role to attract more alveolar macrophages into the airways (Borges et al., 2001Go). Additional studies will be required to better understand the mechanisms of particle-induced apoptosis and its role in cell injury.


    ACKNOWLEDGMENTS
 
This work was supported by NIH HL 10360 and NIH ES 09433.


    NOTES
 
1 To whom correspondence should be addressed at Department of Pharmaceutical Sciences, 372 Fairfield Rd., U- 2092, University of Connecticut, Storrs, CT 06269. Fax: (860) 486-4998. E-mail: Andrea.Hubbard{at}uconn.edu. Back


    REFERENCES
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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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