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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
mesothelioma; phosphorylation; crocidolite; inflammation
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 and TGF-
, 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 1012 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 -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 34 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
-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 0500 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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).
|
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.
|
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 -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
-tocopherol alone; this treatment actually increases the level of dually phosphorylated p38.
|
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).
|
|
|
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.
|
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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 -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).
-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
-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 |
---|
GRANTS
Financial support for this study was provided by the Colt Foundation.
![]() |
FOOTNOTES |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |