Activation of p38 MAP kinase by asbestos in rat mesothelial cells is mediated by oxidative stress

William A. Swain,1 Kenneth J. O'Byrne,1 and Stephen P. Faux2

1Department of Oncology, University of Leicester, Leicestershire; and 2ELEGI, Napier University, Edinburgh, United Kingdom

Submitted 23 May 2003 ; accepted in final form 12 November 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Asbestos fibers are biopersistent particles that are capable of stimulating chronic inflammatory responses in the pleura of exposed individuals. Exposure of pleural mesothelial cells, the progenitor cell of malignant mesothelioma, to asbestos induces an array of cellular responses. The present studies investigated whether the p38 mitogen-activated protein kinase cascade was induced under asbestos-exposed conditions. p38 plays a vital role in the response to stressful stimuli and enables the cell to enter an inflammatory state characterized by cytokine production. Western blot and in vitro kinase assays showed increases in dual phosphorylation and actual activity of p38 after exposure to fibrous and nonfibrous (milled) crocidolite; in contrast, polystyrene beads and iron (III) oxide had no such effects. In common with other asbestos-induced events, this was shown to be an oxidative stress-sensitive effect, inasmuch as preincubation with N-acetyl-L-cysteine or {alpha}-tocopherol (vitamin E) ameliorated the effect. The present studies show that p38 activity is important for crocidolite-induced activator protein-1 DNA binding, inasmuch as an inhibitor of p38, SB-203580, reduced this activity. Crocidolite-induced cytotoxicity was also reduced with SB-203580, indicating a role for p38 in asbestos-mediated cell death. Our studies suggest that p38 activity could be a crucial factor in the chronic immune response elicited by asbestos and may represent a target for future pharmacological intervention.

mesothelioma; phosphorylation; crocidolite; inflammation


EXPOSURE TO ASBESTOS FIBERS, a group of crystalline silicates, is the causative agent associated with the development of malignant pleural mesothelioma, an invariably fatal tumor arising from the mesothelial lining of the pleura (33). The molecular events that precede the development of mesothelioma by asbestos are unclear, but the activation of cell-signaling cascades at the plasma membrane may lead to alterations in gene expression, contributing to chronic inflammation and unregulated mesothelial cell proliferation (24, 25). It is thought that a number of crucial changes are required in a population of mesothelial cells in order for them to become transformed (21). These changes may arise by the direct interaction of the fibers with cells or by a more indirect mechanism whereby the presence of fibers elsewhere in the lung triggers a plethora of chronic and acute inflammatory responses mediated by cells of the pulmonary immune system (16). In reality, a combination of these factors probably initiates the pathogenic events leading to mesothelioma.

Recently, research has focused on intracellular signaling pathways that may contribute to the initiation and progression of the disease process (24). Mitogen-activated protein kinases (MAPKs) are important regulatory proteins that are involved in the phosphorylation of a number of proteins and transcription factors. Moreover, MAPK signaling is linked to transcriptional activation of genes linked to key cellular events such as apoptosis, cell proliferation, differentiation, and development (1). There are three arms of the MAPK signaling cascade: the extracellular-regulated kinases (ERKs), the c-Jun NH2-terminal kinases (JNKs), stress-activated protein kinase (SAPKs), and p38 kinase. They are described as consisting of a module of three enzymes. The first is MAPK kinase kinase, which phosphorylates and activates the second member, MAPK kinase, which in turn activates the canonical member of the pathway, MAPK. Previous studies have shown that asbestos fibers are able to modulate a number of intracellular signaling pathways, including the ERK cascade in primary rat pleural mesothelial cells (24, 36). Stimulation of the ERK cascade has been shown to be involved in fiber-induced apoptosis in this cell type (14, 37). Activation of ERK by asbestos in mesothelial cells is mediated through phosphorylation of the epidermal growth factor receptor (EGFR) via generation of reactive oxygen species (ROS), inasmuch as EGFR tyrosine kinase inhibitors and antioxidants such as N-acetyl-L-cysteine (NAC) are capable of blocking this response.

In contrast to ERK, the JNK pathway is unaffected by asbestos under the same conditions in mesothelial cells (14). Investigation of the p38 pathway under asbestos-stimulated conditions has resulted in somewhat contradictory observations. Geist et al. (6) showed that asbestos could induce p38 kinase activity in human alveolar macrophages after exposure to amosite asbestos (6), whereas Ding and coworkers (2) found that crocidolite did not activate p38 in the mouse epidermal cell line JB6 P+. However, no published studies have analyzed the effects of asbestos on p38 MAPK activation in mesothelial cells.

The p38 and JNK pathways are collectively termed SAPKs because of their role in the way cells meter their response to cytotoxic stimuli such as UV irradiation, proinflammatory cytokines, and osmotic shock (18). These pathways are linked to cytokine signaling, in particular IL-1{beta} and TGF-{beta}, which suggests that p38 is involved in inflammation and ensuing tissue repair. Therefore, a growing body of evidence suggests that p38 may be a good target in combating diseases associated with chronic inflammation (9). Many p38-dependent effects may be mediated through modulation of activator protein-1 (AP-1), a transcription factor composed of Jun:Jun homodimers or Fos:Jun heterodimers (19). p38 lies upstream of Jun expression through its ability to potentiate the transcriptional activity of activating transcription factor-2, which in turn can lead to upregulation of Jun protein (22). The effect of crocidolite fibers on the regulation of AP-1 is well documented (3, 11, 30), and we aimed to test whether these increases were, in part, dependent on the p38 pathway.

In the present studies, we have investigated the mechanism of AP-1 regulation in a rat mesothelial cell line, 4/4 RM-4, by asbestos and determined whether this involves activation of p38 MAPK. Our results suggest that p38 is activated in mesothelial cells by asbestos via a mechanism involving oxidative stress and that this response is involved in the regulation of AP-1 in these cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials. All chemicals were obtained from Sigma unless otherwise stated.

Cell culture and growth medium. A rat pleural mesothelial cell line, 4/4 RM-4, was obtained from the European Collection of Animal Cell Cultures (Porton Down, Wiltshire, UK) and grown in Ham's F-12 medium (GIBCO) containing 15% fetal calf serum, 50 U/ml penicillin, and 100 µg/ml streptomycin. The cells were grown to ~90% confluency in 100-mm dishes, and the fetal calf serum was reduced to 0% 24 h before treatments with test agents.

Particles. Reference samples of crocidolite asbestos were obtained from the Union Internationale Contre le Cancer (UICC). Milled crocidolite was generated in an agate ball mill for 8 h. To ensure that no fibrous particles were present, samples were viewed under electron microscopy. After sterilization in a bench-top autoclave, fibers were suspended in medium at 2 mg/ml and triturated 10–12 times through a 22-gauge needle, and the appropriate volume of this suspension was added to cells to achieve the desired fiber concentration. Polystyrene beads with a mean diameter of 0.2 µm were obtained from Polysciences (Park Scientific, Northampton, UK).

Chelation of Fe2+/Fe3+ from particles. Samples were suspended in a 2 mM ferrozine solution for 24 h on a rotating platform for removal of Fe2+. They were then washed in PBS and resuspended in 2 mM deferoxamine for a further 24 h for removal of Fe3+. Ferrozine/deferoxamine solutions were retained for calculation of the total mass of bioavailable iron. Absorbances for Fe2+/ferrozine and Fe3+/deferoxamine were read at 562 and 430 nm, respectively. Standard solutions of FeSO4/ferrozine and FeCl3/deferoxamine were used to construct standard curves for calculation of the iron content of the chelated samples.

Immunoprecipitation and kinase assays. Cells were lysed in 1 ml of Triton lysis buffer (20 mM Tris, pH 7.4, 137 mM NaCl, 25 mM {beta}-glycerophosphate, 2 mM sodium pyrophosphate, 2 mM EDTA, 10% glycerol, 2 mM benzamidine, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml pepstatin, 5 µg/ml aprotonin, 5 µg/ml leupeptin, 1% Triton X-100, and 0.5 mM dithiothreitol) and then incubated on ice for 15 min before storage at -80°C if required. For immunoprecipitation, 5 µl of p38 antibody diluted to 600 µg/ml were added to 20 µl of protein A-agarose beads and incubated at room temperature for 1 h with occasional flicking; then the beads were washed with PBS to remove unbound antibody. Lysates were centrifuged at 13,000 rpm for 15 min to remove cellular debris; then the supernatant was added to the bead-antibody complex and tumbled for 3–4 h at 4°C. The immune complexes were washed twice in Triton lysis buffer and once in kinase buffer (25 mM HEPES, pH 7.4, 25 mM {beta}-glycerophosphate, 25 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, and 0.5 mM sodium orthovanadate) and then incubated with 30 µl of kinase buffer, 2 µCi of [32P]ATP (Amersham), 3 µl of 835 µM cold ATP (to reach a final ATP concentration of 50 µM), and 5 µg of phosphorylated heat- and acid-stable protein regulated by insulin (PHAS-I; Calbiochem, La Jolla, CA) substrate for 30 min at 30°C. Reactions were stopped by the addition of Laemmli buffer and then heated at 100°C for 5 min. Finally, samples were spun for 30 s and resolved by 13% SDS-PAGE. Incorporation of 32P into the substrate was measured using a beta imaging system (Molecular Dynamics).

Isolation of nuclear proteins and electrophoretic mobility shift assays of AP-1 DNA binding. Cells were treated with crocidolite asbestos (25 µg/cm2) for 24 h in the presence or absence of a 2-h preincubation with SB-203580, a potent and selective inhibitor of p38 (20). Nuclear extracts were isolated and analyzed as described by Mossman and Sesko (25).

Western blotting. Samples that had been analyzed and corrected for protein concentration were resolved by 10% SDS-PAGE, transferred to a nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany), and blocked for 2 h at room temperature with TBS (50 mM Tris, pH 8.0, 150 mM NaCl), 0.1% Tween 20 (TBS-T), and 10% nonfat dry milk. Membranes were incubated for 2 h at room temperature with p38 primary antibody (Sigma) in TBS-T and 5% milk or overnight at 4°C with p38 dually phosphorylated at Thr180 and Tyr182 primary antibody (New England Biolabs, Beverly, MA) in TBS-T and 5% BSA. After they were washed in TBS-T, membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. After the membranes were washed again with TBS-T, the enhanced chemiluminescence system (Amersham) was used to detect protein levels.

Effect of SB-203580 on the toxicity of crocidolite in 4/4 RM-4 cells. 4/4 RM-4 cells were seeded into 96-well plates at 3 x 104 cells/well and allowed to settle overnight before serum starvation for 24 h. They were then pretreated with 0–500 nM SB-203580 for 2 h before incubation in the presence or absence of crocidolite (25 µg/cm2) for a further 24 h. DMSO was included as a vehicle control. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay according to the method of Mossman (23). Briefly, after incubation with test agents, the medium was replaced with complete medium containing MTT. The cells were then incubated for 1 h in the presence of MTT at 37°C. The medium containing MTT was replaced with DMSO to solubilize the formazan product, and the plates were agitated to ensure complete solubilization of the dye product. The absorbance of each sample was then assessed at 540 nm. In each experiment, untreated control cells were used to set the cell viability at 100%.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of crocidolite asbestos on p38/phosphorylated p38 levels in 4/4 RM-4 cells. Levels of p38 dually phosphorylated at Thr180 and Tyr182 were examined after the fibers were exposed for 24 h to 1.2 mM anisomycin employed as a positive control. Asbestos induced significant (P < 0.05) dose-dependent increases in phosphorylated p38 protein at this time point (Fig. 1A), with maximum levels reaching 1.5 times control values. Blots were stripped and reprobed for native p38 levels. Regardless of treatment, expression of p38 remained unchanged, indicating that loading of protein was equal and that the rises in phosphorylated p38 levels were proportional to overall p38. To test the specificity of this response to fibers, two control particles were used. Polystyrene beads (0.2 µm) and iron (III) oxide were added to cells at doses that had elicited responses with fibers. Neither of these particulates was able to induce phosphorylation of p38 at any of the doses tested (Fig. 1B).



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Fig. 1. Effect of particles on p38 phosphorylation in mesothelial cells. 4/4 RM-4 cells were grown to 90% confluency, serum starved for 24 h, and then treated with crocidolite asbestos (A) or polystyrene beads or iron (III) oxide (B) for 24 h or 1.2 mM anisomycin (Aniso) for 30 min. Cells were then harvested and lysed. In A, protein samples (50 µg) were separated by SDS-PAGE and analyzed by immunoblotting with an antibody specific for dually phosphorylated p38 at Thr180 and Tyr182. The same samples were used to probe for native p38 to show equal loading of protein. Blots are representative of 3 experiments. Blots were scanned using molecular dynamics densitometer, and intensity of band relative to control is shown. pp38, phosphorylated p38. Values are means ± SE; n = 3. *P < 0.05.

 

Additionally, the time course of p38 phosphorylation was investigated by probing phosphorylated p38 levels at 4, 8, and 24 h after fiber treatments with untreated control samples taken at each time point to allow for fluctuations in background phosphorylated p38. Crocidolite significantly (P < 0.05) increased phosphorylated p38 levels over the latter two time periods; however, at 4 h these levels were essentially the same as in control incubations (Fig. 2). This lag period may be explained by the time required for fibers to settle onto the cell monolayer. Levels of phosphorylated p38 in untreated controls also rose over the same period, and this is most likely explained by the extended presence of the cells in serum-free medium, which is, in itself, a stressful stimulus. For this reason, we could not further extend the time course, inasmuch as the signal-to-noise ratio became too low (data not shown).



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Fig. 2. Time course of crocidolite-induced p38 phosphorylation. 4/4 RM-4 cells were grown to 90% confluency, serum starved for 24 h, and then treated with 25 µg/cm2 crocidolite asbestos (Croc) for 4, 8, or 24 h, with untreated samples also collected at each time point. Cells were then harvested and lysed. Protein samples (50 µg) were separated by SDS-PAGE and analyzed by immunoblotting with an antibody specific for dually phosphorylated p38 at Thr180 and Tyr182. The same samples were used to probe for native p38 to show equal loading of protein. Blots are representative of 3 experiments and were analyzed as described in Fig. 1 legend. Values are means ± SE; n = 3. *P < 0.05.

 

Effect of UICC crocidolite on p38 kinase activity in 4/4 RM-4 cells. In vitro complex kinase assays are a direct way to measure the actual p38 kinase activity in cells. First, total p38 MAP kinase is immunoprecipitated from cell lysates and then used to radiolabel a peptide substrate (PHAS-I). The proportion of active p38 in the sample will be represented by the relative intensity of phospho-PHAS-I bands.

Figure 3 shows how the activity of p38 is altered with increasing doses of UICC crocidolite and also with the known inducer of p38 activity, anisomycin. Activity of p38 with 5 µg/cm2 crocidolite is essentially the same as p38 activity of the untreated control. At higher doses (10 and 25 µg/cm2), however, p38 activity is increased to nearly double that of control. Indeed, this level of induction is very similar to that observed in the analysis of phosphorylated p38 by Western blotting under identical exposure conditions.



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Fig. 3. Effect of crocidolite on p38 kinase activity in 4/4 RM-4 cells. 4/4 RM-4 cells were grown to 90% confluency, serum starved for 24 h, and then treated with 5, 10, or 25 µg/cm2 Union Internationale Contre le Cancer crocidolite for 24 h or 1.2 mM anisomycin for 30 min. Cells were then harvested and lysed. Total p38 was immunoprecipitated and used in an immune complex kinase assay to radiolabel the phosphorylated heat- and acid-stable protein regulated by insulin (PHAS-I) substrate. Phosphorylated substrate was detected after SDS-PAGE by autoradiography. Blots are representative of 3 experiments and were analyzed as described in Fig. 1 legend. Values are means ± SE; n = 3. *P < 0.05.

 

Mechanism of p38 activation by crocidolite in 4/4 RM-4 cells. The importance of oxidants in the crocidolite-induced activation of p38 was investigated by preincubation of the cells with 1 and 5 mM NAC for 18 h before exposure to 25 µg/cm2 crocidolite for a further 24 h. In common with crocidolite-induced ERK activation (14), NAC blocked the phosphorylation of p38 in a dose-dependent manner, where coincubation with 5 mM NAC returned phosphorylated p38 to untreated control levels (Fig. 4A). To further elucidate the oxidant mechanism involved in p38 activation, cells were incubated for 2 h with 0.5 and 1 mM {alpha}-tocopherol, an inhibitor of lipid peroxidation that we had hypothesized may be important in the activation of p38 by asbestos. Figure 4B shows that these doses decrease asbestos-induced p38 phosphorylation to near-control levels in a dose-dependent manner. A further observation worthy of note is the effect of 1 mM {alpha}-tocopherol alone; this treatment actually increases the level of dually phosphorylated p38.



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Fig. 4. Mechanism of crocidolite-induced p38 phosphorylation. 4/4 RM-4 cells were grown to 90% confluency and serum starved for 6 h before addition of N-acetyl-L-cysteine (NAC) to the medium to reach the final doses indicated (1 or 5 mM) for a further 18 h (A), or vitamin E (0.5 or 1 mM) was added 2 h before fiber exposure (B). Where appropriate, fibers were added to the dish with 25 µg crocidolite/cm2 for 24 h. Blots are representative of 3 experiments and were analyzed as described in Fig. 1 legend. Values are means ± SE; n = 3. *P < 0.05 vs. untreated control; **P < 0.05 vs. crocidolite alone.

 

The data suggest that p38 activation by crocidolite is dependent on the formation of lipid peroxides at the plasma membrane, and this theory is strengthened by observations in other laboratories that crocidolite is capable of inducing lipid peroxidation in mesothelial cells under fiber-stressed conditions (34). Whether this activation is direct or via another signaling intermediary remains to be elucidated.

To examine whether the p38 response is fiber specific, mesothelial cells were exposed to comparable concentrations (mass per unit area) of milled crocidolite, which enables crude dissection of the chemical and physical properties of these fibers. The milled sample did, indeed, cause large increases in phosphorylated p38 over sham-treated controls (Fig. 5). Additionally, these increases were larger than the response seen with comparable mass-per-unit area doses of fibrous crocidolite, and we concluded that this response was due to the increased surface area of the milled sample compared with the fibers on a mass-per-mass basis and could therefore be explained by nonspecific effects of bioavailable iron on the mineral particle surface. To test this hypothesis, we chelated the iron from milled and fibrous crocidolite samples. As shown in Table 1, the milled sample yielded over three times as much bioavailable iron as the fibrous sample. When we exposed cells to the chelated fiber sample, the level of phosphorylated p38 was reduced compared with sham-chelated fibers, although it was still higher than in untreated controls. However, chelation of the milled sample completely abolished the ability of the preparation to induce p38 phosphorylation above untreated control levels (Fig. 6).



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Fig. 5. Dose-dependent increases in phosphorylated p38 by milled crocidolite. 4/4 RM-4 cells were treated with comparable doses (mass per unit area) of milled nonfibrous crocidolite for 24 h or 1.2 mM anisomycin for 30 min. Cells were then harvested and lysed. Protein samples (50 µg) were separated by SDS-PAGE and analyzed by immunoblotting with an antibody specific for dually phosphorylated p38 at Thr180 and Tyr182. Blots are representative of 3 experiments and were analyzed as described in Fig. 1 legend. Values are means ± SE; n = 3. *P < 0.05.

 

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Table 1. Levels of iron chelated from milled and fibrous UICC crocidolite

 


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Fig. 6. Fiber geometry is important for p38 phosphorylation. Fiber preparations were chelated as required (see MATERIALS AND METHODS) before exposure to cells as described in Fig. 5 legend. C Croc, chelated crocidolite; C Milled, chelated milled crocidolite. Blot is representative of 3 experiments.

 

Effect of the p38 inhibitor SB-203580 on crocidolite-induced AP-1 DNA binding. The importance of p38 activation on downstream effectors is illustrated in its ability to phosphorylate a number of proteins, e.g., activating transcription factor-2 and c-Jun, which can dimerize to form AP-1 complexes, which are capable of binding to 12-O-tetradecanoylphorbol-13-acetate-responsive element sites found in the promoter region of genes involved in many cellular processes (17). Phosphorylation of these proteins may be important in transactivation of these transcription factors, enabling the potentiation of a transcription-mediated response independently of de novo protein synthesis. Crocidolite has previously been implicated in upregulation of AP-1 DNA binding (3, 4, 8), and the present studies investigated whether p38 activation contributed to this response. Concentrations of crocidolite known to activate AP-1 (4) were used in cells preincubated for 2 h with SB-203580, a well-documented inhibitor of p38, to determine whether this effect could be ameliorated.

The level of crocidolite-induced AP-1 DNA binding was reduced when cells were treated with 250 or 500 nM SB-203580 (Fig. 7); the specificity of this effect is shown in the final lanes, where a DMSO vehicle control treatment had no effect on crocidolite-induced AP-1 DNA binding. The present data suggest that in this system the p38 pathway is important in the formation of activated AP-1 complexes, which are capable of binding to DNA; however, it is clear from the present studies and previously published results that p38 is not the sole contributor to AP-1 DNA binding (37). Instead, it seems more plausible that activation of ERK through the EGFR pathway would also contribute via upregulation of c-fos expression through transcription by the serum response factor.



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Fig. 7. p38 activation contributes to crocidolite-induced activator protein-1 (AP-1) DNA binding. 4/4 RM-4 cells were grown to 90% confluency, serum starved for 24 h, and then pretreated with 500 nM SB-203580 (a p38 inhibitor) or DMSO vehicle control for 2 h before addition of 25 µg/cm2 crocidolite for a further 24 h. Cells were harvested, and nuclear proteins were extracted; 4 µg of this extract were incubated with a 32P-labeled AP-1 oligonucleotide and separated by 4% PAGE. Levels of retarded AP-1 were detected by autoradiography. NF-{kappa}B, nuclear factor-{kappa}B. Blot is representative of 3 experiments.

 

Effect of SB-203580 on crocidolite-mediated cytotoxicity. We used MTT assays to assess whether the modulation in p38 activity induced by crocidolite affected phenotypic end points associated with this insult, i.e., cell death. Initial studies with SB-203580 alone indicated that it reduced cell viability ~10% at 100 nM, which was significant (P < 0.05). SB-203580 at 250 or 500 nM had no significant effects on cytotoxicity (Fig. 8A). Crocidolite at 25 µg/cm2 reduced cell viability to ~80%, which is consistent with observations of other groups under these conditions (14, 31). A 2-h preincubation with 250 or 500 nM SB-203580 significantly (P < 0.05) increased cell viability compared with treatment with crocidolite only. However, a return to untreated control levels was not observed (Fig. 8B), suggesting that additional factors are involved in crocidolite-mediated cell death.



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Fig. 8. Modulation of crocidolite-induced cytotoxicity by SB-203580. 4/4 RM-4 cells (3 x 104) were plated in wells of a 96-well plate and left to settle for 24 h before serum starvation for a further 24 h. Crocidolite at 25 µg/cm2 was added in the presence or absence of a 2-h preincubation with 100, 250, and 500 nM SB-203580. Values are means ± SE; n = 3. *P < 0.05 compared with untreated control; **P < 0.05 compared with crocidolite alone.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The molecular mechanisms underlying the pathogenicity of mineral fibers are poorly understood. However, in recent years, several key findings have provided insight into the factors that may be important in the initiation of carcinogenesis. Undoubtedly, there is a role for the ERK cascade, with studies now having mapped out cell signaling routes from the cell surface and culminating in apoptosis (14, 36). In the midst of this relation seems to be the protooncogene c-fos, which has a well-established association with asbestos (8, 12, 31). Studies in other laboratories have shown that the JNK signaling pathway is not activated by asbestos under the same conditions (14) and that activation of p38 may be dependent on cell type (2, 6). In the present studies, we have shown that in a rat mesothelial cell line the second arm of the SAPK cascade, p38 MAPK, is, in fact, activated in a dose-dependent manner. This phenomenon has been shown by the use of Western blots for phosphorylated p38 and also via immune complex kinase assays. The latter technique is a direct measure of the actual activity of total cellular p38 and confirmed the immunoblot data. Given that these techniques do not indicate the localization of phosphorylated p38, it would be useful to perform an immunofluorescence-based analysis to show whether it was just the cells directly in contact with fibers that were exhibiting elevated p38 phosphorylation or a more generalized response. The parameters of p38 activation described above appear to correlate well with the activation of the ERK pathway under similar conditions in this cellular model (36). Our observations were further strengthened after experiments with nonpathogenic control particles. When used at comparable mass per unit area, neither polystyrene beads nor iron (III) oxide was able to mimic the effects of asbestos fibers. This suggests that the p38 response noted in these cells is not merely a nonspecific response to particles/iron but is dependent on an inherent property of the fibers themselves. The mechanisms of p38 activation we have investigated in these studies reflect observations seen throughout the field of asbestos-mediated lung injury and carcinogenesis. The pathogenicity of asbestos fibers appears to rely heavily on the generation of ROS, which may be generated directly from the fiber surface (5) or indirectly through a process of frustrated phagocytosis (16). In the in vitro system employed here, it appears that both could be relevant, inasmuch as chelation of iron from the fiber surface abrogated but did not abolish p38 activation, and the residual levels could be accounted for by the phagocytic response, which would ensue if it is assumed that the physical properties of the fibers were unaltered by the chelation process. However, it is quite possible that not all the iron was chelated, and residual levels may be responsible for this observation.

Although the exact source of ROS may be slightly unclear, the importance of ROS in the activation of p38 is not, inasmuch as NAC and {alpha}-tocopherol could completely abrogate its activation. NAC is a cell membrane-permeable precursor of glutathione, which is a major substrate for antioxidant enzymes in eukaryotic cells (15), allowing detoxification of peroxide species such as H2O2. Indeed, a number of these enzymes have previously been shown to be upregulated by asbestos (13). {alpha}-Tocopherol is a more specialized antioxidant because of its lipophilic nature. It is found in cell membranes and lipoproteins and specifically acts to neutralize lipid peroxides, which may trigger oxidant-producing chain reactions (7). We recently showed increases in one of these lipid peroxides, 4-hydroxynonenal, in asbestos-exposed mesothelial cells (unpublished observations). This may represent a possible mechanism whereby the stimulus from extracellular fibers may be transmitted to intracellular responses, inasmuch as 4-hydroxynonenal can act as a signaling intermediary capable of activating stress-signaling pathways such as p38 (32). It cannot be discounted that receptor-mediated events lie upstream of p38 activation, however, inasmuch as this is a common feature in other cellular systems where stress-induced activation of p38 occurs (10, 35). The finding that {alpha}-tocopherol alone induces p38 phosphorylation may be due to a phenomenon known as tocopherol-mediated peroxidation. This term describes a process where this vitamin does not act as a chain-breaking antioxidant; instead, it exhibits prooxidant behavior, which could allow activation of p38, perhaps via a mechanism similar to that outlined above (29).

In the present studies, we have also examined the downstream effect of p38 activation on crocidolite-induced AP-1 DNA binding, a phenomenon that has previously been well reported (2, 8). The AP-1 transcription factor is extremely well understood, being composed of Jun:Jun homodimers or Jun: Fos heterodimers. The exact composition may influence which subset of genes is targeted for transcription by the complex (19). Given that there are multiple members of the Fos and Jun protein family, this may account for the pleiotropic effects of AP-1 activation. Recently, experimental evidence suggests that one member of the Fos family, fra-1, is critical for asbestos-induced transformation of mesothelial cells. Moreover, this study detected fra-1 at increased levels in mesothelioma cell lines, and transfection with dominant-negative fra-1 constructs reversed their malignant phenotype (27). In our system, the activation of p38 by crocidolite may contribute to the prolonged AP-1 response noted under these conditions, which in turn may contribute to the phenotypic end points reached in mesothelial cells exposed to asbestos. We have examined a role for p38 in reaching these end points by using the MTT assay, and the present studies have shown that SB-203580 had a small but significant effect when added alone (P < 0.05) at the lowest dose tested (100 nM), but the relevance of this effect is uncertain. The cytotoxicity of crocidolite is well defined, however, and we noted a decrease in cell viability of ~20% with 25 µg/cm2 crocidolite alone. This value is very much in keeping with the work of previous investigators, who showed that asbestos can induce apoptosis in ~20% of cells (14). When p38 was inhibited before asbestos exposure, this effect was reduced significantly (P < 0.05) at 250 and 500 nM SB-203580. This finding suggests that p38 is important for crocidolite-mediated cytotoxicity; however, the exact nature of the cell death cannot be determined from this assay. Despite the limitations of this work, studies from other laboratories suggested that this death probably occurs through apoptosis (26, 28). Additionally, cell viability was not completely restored, which indicates that other factors are involved, and previous work would suggest this to be the case, where, at least in part, the ERK pathway plays a role in crocidolite-induced mesothelial cell cytotoxicity (14). The effects of milled crocidolite on cell viability were not assessed in this study, and this represents an area worthy of future study that could provide further insight into the mechanisms of asbestos-mediated cytotoxicity.

In conclusion, the present studies have demonstrated for the first time that crocidolite can activate the p38 arm of the SAPK pathway in a rat mesothelial cell line and that this lies upstream of crocidolite-mediated AP-1 DNA binding and cell death. Further work in this area is needed to examine whether activation of this pathway is functionally linked to downstream inflammatory processes before it can be identified as a possible clinical target.


    ACKNOWLEDGMENTS
 
We thank Dr. Rodger Duffin for generously supplying the polystyrene beads.

GRANTS

Financial support for this study was provided by the Colt Foundation.


    FOOTNOTES
 

Address for reprint requests and other correspondence: W. A. Swain, Dept. of Oncology, Osborne Bldg., Univ. of Leicester, Leicester, UK (E-mail: was1{at}le.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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