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Asbestos-Induced Apoptosis Is Protein Kinase C-Dependent

Departments of Pathology and Pharmacology, University of Vermont College of Medicine, Burlington, Vermont

Address correspondence to: Brooke T. Mossman, Ph.D., Department of Pathology, University of Vermont College of Medicine, 89 Beaumont Avenue, Burlington, VT 05405. E-mail: brooke.mossman{at}uvm.edu

	Abstract

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Alveolar epithelial and mesothelial cells undergo apoptosis in response to asbestos, a phenomenon that may be important in injury and/or initiation of compensatory proliferation. Here, we report a functional role of protein kinase (PKC) in apoptosis by crocidolite asbestos. We first show that asbestos increases the kinase activity of PKC in alveolar type II epithelial cells (C10 line) and causes its translocation to mitochondria, events associated with caspase-9 cleavage and apoptosis as detected by the Apostain technique. Pretreatment of C10 cells with rottlerin (Rot), a PKC–selective inhibitor, before addition of asbestos prevented cleavage of caspase-9 and blocked the appearance of apoptotic cells. Asbestos-induced apoptosis also was inhibited in cells stably expressing a dominant-negative kinase-deficient mutant of PKC (dnPKC), but not dnPKC. Activities of PKC and PKC increased after exposure to asbestos, but neither isoform migrated to mitochondria. A general inhibitor of PKCs, bisindolylmaleimide I, had no effect on asbestos-induced apoptosis. Hydrogen peroxide (H₂O₂) induced activation of PKCs , , , and , translocation of PKC to mitochondria, and caspase-9 cleavage. However, H₂O₂-induced apoptosis was not inhibited by cell lines stably expressing either dnPKC or dnPKC, suggesting that activation of PKC has a distinct role in the development of asbestos-induced apoptosis.

Abbreviations: bisindolymaleimide, Bis • bovine serum albumin, BSA • diacylglycerol, DAG • dominant negative PKC, dnPKC • fetal bovine serum, FBS • hydrogen peroxide, H₂O₂ • hemagglutinin, HA • mitogen-activated protein kinase, MAPK • phosphate-buffered saline, PBS • PBS containing 0.1% Tween-20, PBST • phorbol dibutyrate, PDBu • protein kinase C, PKC • rottlerin, Rot • room temperature, RT • Tris-buffered saline, TBS

	Introduction

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Asbestos fibers are a chemically and physically diverse group of mineral silicates which are associated with the development of lung cancer, malignant mesothelioma, and fibroproliferative disease, i.e., asbestosis (1). Asbestos induces oxidative DNA damage in cell-free systems as well as in cells in vitro (2, 3). In addition to mutagenesis and/or transformation, DNA damage also results in necrosis and/or apoptosis, which may be responsible for the resolution of alveolar type II cell hyperplasia in acute lung injury (4). Asbestos causes apoptosis in mesothelial and alveolar epithelial cells, and this process may involve generation of reactive oxygen species and activation of mitogen-activated protein kinases (MAPKs) (5–8).

In this report, we focused on the protein kinase C (PKC) family of enzymes as potential upstream activators of MAPKs and regulators of asbestos-induced apoptosis in murine pulmonary epithelial type II cells (C10). The PKC family consists of 11 isoforms, whose expression varies among cell types (9). Individual isoforms exhibit varying substrate specificity, as well as differences in their subcellular localization and response to specific stimuli (9, 10). A variety of studies indicate that specific isoforms of PKC may be either pro-apoptotic or anti-apoptotic, depending upon the stimulus and cell type (11, 12).

PKC is involved in asbestos-induced proto-oncogene (fos/jun) expression in mesothelial cells, and downregulation or inhibition of PKC prevents asbestos-induced proto-oncogene expression (13). Recently, we have also reported that PKC is activated in C10 cells exposed to asbestos or after wounding, and is elicited in pulmonary epithelial cells after inhalation of asbestos (14). Although PKC has been shown to be activated by asbestos, its role in asbestos-induced responses remains undefined.

The present studies were undertaken to determine if specific isoforms of PKC regulate apoptosis in pulmonary epithelial cells in response to asbestos. These studies demonstrate that PKC is activated and translocated to mitochondria in response to asbestos, and that the activity of this isoform is essential for the development of asbestos-associated apoptosis. PKC and PKC are likewise activated following treatment of epithelial cells with asbestos; however, neither of these two isoforms migrate to mitochondria following asbestos exposure or are causally involved in apoptosis. Because apoptosis by asbestos is linked to oxidative stress (6, 15), we also examined possible causal associations of PKCs with H₂O₂-induced apoptosis. Results suggest that PKC may have a distinct function in asbestos-induced cell death through mediation of a mitochondrial pathway, whereas H₂O₂-induced apoptosis may involve other mechanisms.

	Materials and Methods

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Chemicals and Antibodies
Polyclonal antibodies to PKC , , , , and caspase-9 were obtained from Santa Cruz (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Phorbol dibutyrate (PDBu) was purchased from Sigma (Sigma Chem. Co., St. Louis, MO). MitoTracker Red 580 was purchased from Molecular Probes, Inc. (Eugene, OR). The protein kinase inhibitors, rottlerin (Rot) and bisindolylmaleimide I (Bis), were obtained from Calbiochem (La Jolla, CA). Monoclonal antibody to single stranded DNA, Apostain (F7–26), was purchased from Alexis (San Diego, CA). Crocidolite asbestos fibers were obtained as reference sample from NIEHS and have been characterized previously (7).

Cell Culture
A contact inhibited, nontransformed murine alveolar type II epithelial cell line (C10) (16) was propagated in CMRL-1066 medium containing penicillin, streptomycin, L-glutamine, and 10% fetal bovine serum (FBS) (GIBCO BRL, Grand Island, NY). For all experiments, cells were grown to near confluence, complete medium was removed, and medium containing 0.5% FBS was added 24 h before exposure to agents. H₂O₂ (Sigma) was added directly to the medium at 300 µM, a concentration known to cause apoptosis (17). Crocidolite asbestos fibers were suspended in Hanks' Balanced Salt Solution (GIBCO BRL) (1 mg/ml), triturated 10x through a 22-gauge needle to obtain a homogenous suspension, and added directly to the medium for a concentration of 5 µg/cm² culture dish. This concentration induces apoptosis at 24 h followed by compensatory proliferation at 72 h (17). Protein kinase inhibitors were added 30 min before asbestos treatment at concentrations used by others to inhibit PKCs (18, 19) (Rot 15 µM; Bis 5 µM). Sham control cultures received medium without agents and were treated identically. Groups in all experiments consisted of two or three determinations per time point, and all experiments were repeated at least twice.

Western Blotting
Cells grown in 100-mm culture dishes were washed three times with ice-cold phosphate buffered-saline (PBS) and collected in lysis buffer (20 mM Tris pH 7.6, 1% Triton X-100, 137 mM NaCl, 2 mM EDTA, 1 mM Na₃O₄V, 10 mM NaF, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) and were incubated on ice for 30 min (17). Cells were then sonicated and centrifuged at 14,000 x g for 15 min at 4°C. Supernatants were collected, and protein concentrations were determined using the Bradford assay (Bio-Rad, Richmond, CA). Cell lysates (20–40 µg) were resolved by SDS-PAGE and transferred to nitrocellulose membranes according to standard procedures. Equal loading of protein was verified by the Ponceau stain (Sigma). Membranes were washed in Tris-buffered saline (TBS), and blocked for 30 min with TBS containing 5% nonfat milk, then incubated with primary antibodies at 1:250 dilution in TBS containing 1% bovine serum albumin (BSA) overnight at 4°C. Membranes were then washed twice with TBS alone and twice with PBS containing 0.1% Tween-20 (PBST) before incubation with horseradish peroxidase–conjugated secondary antibody (1:5,000 in PBST containing 5% nonfat milk) for 1 h at room temperature (RT). Membranes were washed once with PBST and three times with PBS before antibody binding was visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer's protocol.

In Vitro Kinase Activity Assays
PKC, PKC, PKC, and PKC enzymatic activities were assessed using an immunoprecipitation kinase assay as follows. Soluble protein was prepared as described above with some modifications (no sonication and addition of 2 mM pyrophosphate and 25 mM ß-glycerophosphate to the lysis buffer). The protein (300 µg) then was immunoprecipitated for 4 h at 4°C using 2 µg of anti-PKC, anti-PKC, anti-PKC, or anti-PKC antibody. The antigen–antibody complexes were collected by incubation with Agarose–protein A (GIBCO BRL) for 1 h at 4°C, then pellets were washed three times with lysis buffer and three times with kinase buffer (20 mM Tris pH 7.4, 10 mM MgCl₂, 10 µM ATP, and 1.25 mM CaCl₂) and resuspended in a reaction buffer containing 25 µl kinase buffer, 0.25 mg histone (Sigma), and 5 µCi of [ ³²P]ATP (New England Nuclear, Life Science Products Inc., Boston, MA). PKC isoforms requiring lipids were incubated in a reaction buffer containing 50 µg/ml phosphatidylserine and 4.1 µM dioleoylglycerol. All samples were incubated for 20 min at 30°C. Reactions were terminated by the addition of 2x SDS sample buffer, boiled, and the reaction products resolved on a 12.5% SDS-polyacrylamide gel. The extent of histone phosphorylation was determined by auto-radiography and quantitated using a Phosphor Imager (Molecular Dynamics, Amersham Biosciences, Piscataway, NJ).

Subcellular Fractionation
To isolate the mitochondria-enriched fraction, cells were washed with PBS, then collected in buffer containing 25 mM Tris (pH 7.4), 250 mM sucrose, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT) and protease inhibitors as described above before homogenization 10 times with a Dounce homogenizer. Unlysed cells and nuclei were removed by centrifugation at 750 x g for 10 min. The supernatant was centrifuged at 10,000 x g for 30 min, and the resulting mitochondrial-enriched pellet was washed twice with the same buffer. The supernatant was further spun at 100,000 x g for 1 h to obtain the supernatant, cytosolic fraction. All centrifugations were performed at 4°C. The protein concentration in each sample was then determined by the Bradford method.

Confocal Laser Scanning Microscopy
Cell monolayers grown on coverslips were exposed to crocidolite asbestos (5 µg/cm²) or PDBu (100 ng/ml) for the indicated times. Medium was then replaced with prewarmed media containing MitoTracker Red 580 at 200 nM. Dishes were incubated in the dark at 37°C for 30 min, then washed twice with PBS and fixed in 3.7% paraformaldehyde (in PBS) for 5 min at RT. Cells were then permeabilized in chilled methanol for 3 min at RT. After washing with PBS, the cells were incubated with blocking solution containing 1% BSA/PBS for 1 h at RT. After blocking, primary antibody (rabbit polyclonal PKC antibody, 3 µg/ml; Santa Cruz) diluted in 1% BSA/PBS, was added to the cells for 1 h at RT. Cells were then washed in PBS, and secondary antibody (Alexa Fluor 488 goat-antirabbit IgG, 1:400; Molecular Probes) was applied. Finally cells were washed in PBS, and coverslips were mounted onto slides with AquaPolyMount (Polyscience, Inc, Warrington, PA). For each sample, confocal images were collected in fluorescence modes, followed by electronic merging of the images using a confocal microscope (MRC1024ES; BioRad, Hercules, CA).

Detection and Quantitation of Apoptosis
For detection of apoptosis, cell monolayers grown on glass coverslips were exposed to agents as described earlier, then fixed in 100% methanol at –20°C for 24 h. To induce DNA denaturation in situ, cells were heated to 100°C in PBS containing 5 mM MgCl₂ for 5 min, then immersed in ice-cold water for 10 min. After incubation with 40% FBS in PBS on ice for 15 min, cells were incubated with a monoclonal antibody to single-stranded DNA (10 µg/ml, Apostain F7–26) for 30 min at RT, then washed twice in PBS and incubated with horseradish peroxidase–conjugated secondary antibody (15 µg/ml, goat antimouse IgM; Jackson Laboratories, West Grove, PA) for 30 min at RT. To visualize secondary antibody binding, the peroxidase substrate DAB (Sigma) was used. Cells were washed and mounted on slides in 50% glycerol in PBS for subsequent examination using bright field light microscopy. To determine the numbers of apoptotic cells and total cell numbers per field, 10 random fields were evaluated at x400 magnification on duplicate coverslips. Apoptosis by asbestos was confirmed by transmission electron microscopy as described previously (17).

Creation of Stable C10 Lines Expressing Dominant-Negative PKC and PKC
C10 cells were transfected with full-length mouse PKC with a K376R point mutation (dnPKCs) or PKC with a K368R point mutation (dnPKC) at the ATP-binding site, cloned into the hemagglutinin (HA)-tagged, neomycin-resistant plasmid, pcDNA3HA (a kind gift from Dr. Arshad Rahman, University of Illinois at Chicago, obtained originally from the lab of Dr. Bernard Weinstein, Columbia University, NY). Control cells were transfected with an empty vector. The cells were transfected using Lipofectamine 2,000 (Invitrogen, Life Technologies, Grand Island, NY) according to the manufacturer's specifications, and transfected clones were identified based upon growth in Geneticin containing medium over 14 d (500 µg/ml; Cellgro; Mediatech, Inc., Herndon, VA). Controls consisted of cells transfected with the empty vector having the geneticin-resistant gene, but without mutated PKC or . Expression of mutated PKC or protein in the transfected cell lines was confirmed by immunofluorescence of HA-tagged protein using an anti-HA high-affinity antibody (rat monoclonal; Roche, Mannheim, Germany).

Statistical Analysis
Results were evaluated by one-way ANOVA using the Student-Newman-Keuls procedure for adjustment of multiple comparisons. Differences with P values 0.05 were considered statistically significant.

	Results

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Changes in the Cytosolic Expression of Different PKC Isoforms Occur during Asbestos Exposure
We have previously shown that crocidolite asbestos (5 µg/cm²) induces apoptosis in C10 cells over a 24-h period (17). As an initial approach, we asked if exposure of C10 cells to asbestos resulted in changes in the abundance of specific PKC isoforms over time. We selected four isoforms representative of three different PKC classifications; i.e., PKC = conventional, PKC and = novel, and PKC = atypical. Figure 1 shows an immunoblot of untreated C10 cells and those exposed to asbestos (5 µg/cm², 8 or 16 h), H₂O₂ (300 µM, 8 h), or the phorbol ester, PDBu (100 ng/ml, 1 h). H₂O₂ was selected as a positive control causing oxidative stress, and PDBu was a positive control for PKC stimulation. Cytosolic expression of three isoforms of PKC (, , ) increased at 8 h and 16 h in response to apoptotic concentrations of asbestos. Both H₂O₂ and PDBu also increased protein levels of all three isoforms in cytosolic preparations.

View larger version (78K): [in this window] [in a new window]   Figure 1. Changes in cytosolic PKC isoforms following exposures to asbestos. Confluent cultures of murine lung type II epithelial cells (C10) were exposed for the indicated times to 5 µg/cm2 crocidolite asbestos, 300 µM H2O2, or 100 ng/ml PDBu. PKC isoform expression was determined by Western blot analysis as described in MATERIALS AND METHODS. Autoradiogram representative of three independent experiments.

The Activities of PKC, PKC, and PKC Increase during the Development of Asbestos-Induced Apoptosis

View larger version (53K): [in this window] [in a new window]   Figure 2. PKC, PKC, and PKC are activated during asbestos-induced apoptosis. Confluent cultures of C10 cells were exposed for to 5 µg/cm2 crocidolite asbestos or 300 µM H2O2. PKC activity was assayed by an immunoprecipitation kinase assay using cell lysates (0.3 mg protein) prepared as described in MATERIALS AND METHODS. Histone phosphorylation (arrows) was assayed with or without exogenous lipids. The reaction product was separated on a 12.5% SDS-polyacrylamide gel. (A) Autoradiogram of the gel. (B) Quantitation of the same gel using phosphorimaging; *P < 0.05. Representative of three independent experiments performed in duplicate.

View larger version (100K): [in this window] [in a new window]   Figure 3. Translocation of PKC to mitochondria in response to asbestos. C10 cells were exposed to 5 µg/cm2 crocidolite asbestos, 300 µM H2O2, or 100 ng/ml PDBu for the indicated times. (A) Cells were harvested, and mitochondrial-rich fractions were prepared as described in MATERIALS AND METHODS. Proteins were subjected to 7.5% SDS-PAGE and immunoblot analysis was performed using anti-PKC, , or antibodies. (B) Cells were exposed to medium containing the mitochondria-selective permeant dye, MitoTracker Red 580, for 30 min. Cells were then washed, fixed, and stained for PKC. Shown are merged confocal images. The increased yellow-orange color depicts the colocalization of PKC (green) with the mitochondrial-specific dye, MitoTracker Red 580 (red). Asbestos fibers are white as detected by the reflection mode. (C) Same as B, except that instead of PKC, PKC antibodies were used. Representative of more than three experiments.

Inhibition of PKC Suppresses the Cleavage of Caspase-9

View larger version (29K): [in this window] [in a new window]   Figure 4. Inhibition of caspase-9 cleavage by the PKC inhibitor, rottlerin. C10 cells were exposed to 5 µg/cm2 crocidolite asbestos, 300 µM H2O2, or 100 ng/ml PDBu with or without prior treatment with rottlerin (15 µM) for 30 min. Whole cell lysates were subjected to immunoblot analysis using anti caspase-9 antibodies as described in MATERIALS AND METHODS. Representative of more than three independent experiments.

Inhibition of PKC Suppresses Asbestos-Induced Apoptosis

View larger version (58K): [in this window] [in a new window]   Figure 5. Inhibition of PKC blocks appearance of the apoptotic phenotype in asbestos-treated C10 cells. (A) Confluent cultures of C10 cells were exposed for 24 h to 5 µg/cm2 asbestos with or without inclusion of rottlerin (Rot, 15 µM) and bisindolymaleimide (Bis, 5 µM) 30 min before the addition of asbestos. Arrow shows an apoptotic body. (B) Quantitative representation of data. (C) Dose-dependent effect of rottlerin (Rot) on asbestos-induced apoptosis. *P < 0.05 (as compared with control). Representative of four independent experiments.

Inhibition of Asbestos-Induced Apoptosis by a Dominant-Negative PKC Mutant

View larger version (11K): [in this window] [in a new window]   Figure 6. A dominant-negative mutant of PKC inhibits asbestos-induced apoptosis in murine lung epithelial type II cells. C10 cells stably expressing either empty vector (EV), dominant-negative PKC (dnPKC), or dominant-negative PKC (dnPKC) were exposed (+) to asbestos (5 µg/cm2, 24 h), and apoptosis was quantitated in both + and - (untreated) groups as described in MATERIALS AND METHODS; *P < 0.05 (as compared with empty vector group). The percentage of apoptosis in untreated cells was 2–4% and unaffected by EV alone. Representative of three independent experiments.

Inhibition of Asbestos-Induced PKC Translocation to Mitochondria by a Dominant-Negative PKC Mutant

View larger version (185K): [in this window] [in a new window]   Figure 7. Mitochondrial migration of PKC in response to asbestos in cell lines stably expressing dnPKC. C10 cells stably expressing either empty vector (EV) or dominant-negative PKC (dnPKC) were exposed to asbestos (5 µg/cm2) for 24 h. Cells then were exposed to medium containing the mitochondria-selective permeant dye, MitoTracker Red 580, for 30 min. Cells were washed, fixed, and stained for PKC. Shown are merged confocal images. The increased yellow-orange color depicts the colocalization of PKC (green) with the mitochondrial-specific dye, MitoTracker Red 580 (red). Asbestos fibers detected by the reflection mode are white. Representative of three independent experiments.

	Discussion

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A general feature of tumor promoters such as PDBu, TPA, or asbestos is their ability to stimulate PKC activity. Phorbol esters such as TPA and PDBu resemble diacylglycerol in structure, and thus activate PKC directly. In contrast, asbestos is an insoluble fiber that increases hydrolysis of inositol phospholipids and amounts of diacylglycerol (DAG) in respiratory epithelial cells (25). The role of PKC isozymes in asbestos-induced responses is unclear. Prior studies suggest that asbestos-induced stimulation of MAPK pathways results in the activation of fos/jun early response genes and AP-1 activation (8, 17). In support of the involvement of PKCs, it has been shown that after downmodulation of PKC or use of PKC inhibitors, asbestos-induced c-jun and c-fos mRNA levels are diminished in rat pleural mesothelial cells (13).

Different isoforms of PKCs are implicated both in anti- and pro-apoptotic pathways in several cell types. In support of an anti-apoptotic function, PKC inhibitors are potent inducers of apoptosis in hematopoietic and neoplastic cells (26, 27). Recently, PKC has been shown to phosphorylate Bcl-2 in vitro, and overexpression of PKC results in an increased Bcl-2 phosphorylation and suppression of apoptosis in human pre-B REH cells (28). The atypical PKC isoforms, PKC and PKC, likewise protect against apoptosis (29). In support of a pro-apoptotic role for PKCs, activation of PKC by PMA or its overexpression induces apoptosis in prostate carcinoma cells (30).

Here, we show the causal association of PKC in asbestos-induced apoptosis. Indeed, links between TPA-induced apoptosis and mitochondrial dysfunction by PKC have been reported by others (21, 22, 31). Work by Li and coworkers (31) has shown that in keratinocytes exposed to TPA, overexpressed PKC targets mitochondria, causing shape changes and aggregation. This group has also studied PKC-induced interference with the electron transport chain. Antimycin A, an inhibitor of complex III in the respiratory chain, and rotenone, an inhibitor of complex I, substantially decreased TPA-induced cell death at low concentrations, substantiating a cause-and-effect relationship. Also, PKC causes the mitochondrial membrane to lose its membrane potential, as further confirmed by Majumder and colleagues (22). There are several other mechanisms by which PKC could control apoptosis: (i) by caspase-dependent cleavage of PKC (32); (ii) by mitochondrial translocation of PKC and consequent cytochrome c release (21, 22); and/or (iii) by inactivation of DNA protein kinase (DNA-PK), an enzyme that is essential for the repair of double-stranded DNA breaks (33). In support of hypotheses (i) and (ii), active caspase-8 either directly induces procaspase-3 cleavage or induces cleavage indirectly by a mitochondrial amplification pathway requiring BID cleavage, cytochrome C release, and activation of procaspase-9. We did not see caspase-8 or BID cleavage in C10 cells after addition of asbestos (data not shown), but caspase-9 cleavage was observed. Moreover, inhibition of PKC kinase activity inhibited this cleavage. Caspase-9 cleavage might be attributed to cytochrome c release from mitochondria in response to PKC translocation by asbestos (21), in support of the results of Kamp and colleagues, who have demonstrated loss of mitochondrial membrane potential and release of cytochrome by asbestos fibers at 4 h after their addition to alveolar epithelial cells (5).

We also demonstrate increases in the kinase activity of other isoforms of PKC following their exposure to asbestos, but PKC specifically migrates to mitochondria. The specificity of PKC's ability to migrate to mitochondria is still not clear, but the binding of DAG to a specific location on the regulatory domain of PKC may be responsible for targeting PKC to mitochondria (21). Because asbestos increases DAG levels (25), asbestos-induced translocation of PKC to mitochondria may be a critical event initiating asbestos-induced apoptosis, as suggested in Figure 7. The lack of movement of PKC to mitochondria may be explained by the absence of a DAG binding site in its regulatory domain (33). We also did not observe translocation of PKC to mitochondria. Thus, although PKC and PKC are overexpressed and activated in C10 cells by asbestos, these isoforms probably are not required for the observed mitochondrial changes associated with apoptosis.

In our studies using Rot or a dnPKC, we could inhibit the asbestos-induced apoptosis, whereas Bis, a compound which nonspecifically inhibits several isoforms of PKC (18), had no effects. The lack of effect of Bis on asbestos-induced apoptosis may occur because of its general effects on other isoforms of PKC that have opposing effects on the progression of apoptosis (34, 35).

Our data show some parallels between asbestos- and H₂O₂-mediated signaling. For example, it has been reported in other cell types that H₂O₂ and other oxidative stresses cause translocation of PKC to mitochondria (22). Asbestos-induced PKC activation and translocation to mitochondria may be oxidative stress–dependent, as asbestos causes oxidative stress, and asbestos-induced apoptosis is inhibited by antioxidants (6). In support of our findings, Kamp and coworkers recently reported that mitochondria play an important role in asbestos-induced apoptosis which is mediated via iron-derived ROS in A549 cells (20). Mitochondria are initial targets of asbestos-induced DNA damage, which is associated with mitochondrial localization of oxidants. Unlike asbestos, H₂O₂, a more robust oxidant, caused apoptosis that was PKC-independent in C10 cells. The concept that H₂O₂-induced cell injury may involve other cell signaling pathways is supported by several publications (8, 36, 37).

In conclusion, our studies show that asbestos causes increased activity of PKC that is associated causally with apoptosis in pulmonary epithelial cells. Figure 8 presents a hypothetical sequence of events that are indicated from studies here and those reported previously. Asbestos fibers, either by elaboration of oxidants or interaction with the cell membrane, cause increases in diacylglycerol (25), which then activates cytosolic PKC and permits its translocation to mitochondria. The functional significance of PKC translocation to mitochondria is supported by the finding that this event is linked to caspase-9 cleavage and apoptosis through a mitochondrial-dependent pathway. Our prior studies also demonstrate that PKC translocates to nuclei after wounding or exposures to asbestos, where it colocalizes in cells expressing proliferating cell nuclear antigen (14). The dichotomous outcomes of apoptosis and compensatory proliferation by asbestos are supported by studies in mesothelial cells, showing that early apoptosis causes later increases in DNA synthesis, both processes existing in a dynamic balance at 24 h (38). These findings, and those indicating that PKC inhibition blocks the expression of AP-1 family members linked to proliferation of epithelial cells (14), support an intriguing model whereby asbestos induces early apoptosis via a PKC mitochondrial targeted pathway and compensatory proliferation via a pathway involving nuclear or membrane translocation of PKC. The dissection of these cell-signaling events should have a major impact on preventive and therapeutic approaches for asbestos-associated lung cancers and asbestosis.

View larger version (61K): [in this window] [in a new window]   Figure 8. A model depicting the role of PKC in asbestos-induced apoptosis and compensatory proliferation. Our data here support a model in which asbestos exposure leads to activation of PKC, which then migrates to mitochondria and induces release of cytochrome C. Cytochrome C promotes caspase-9 cleavage, which ultimately leads to apoptosis. This mitochondrial pathway and nuclear pathways of apoptosis may be counterbalanced by cascades leading to proliferation via membrane and/or nuclear localization of PKC.

	Acknowledgments

Received in original form November 13, 2002

Received in final form February 21, 2003

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