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Published ahead of print on December 30, 2004, doi:10.1165/rcmb.2004-0133OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 436-442, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0133OC

Role of Iron in Inactivation of Epidermal Growth Factor Receptor after Asbestos Treatment of Human Lung and Pleural Target Cells

Aleksander Baldys and Ann E. Aust

Department of Chemistry and Biochemistry, Utah State University, Logan, Utah

Correspondence and requests for reprints should be addressed to Ann E. Aust, Ph.D., Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322-0300. E-mail: AAUST{at}cc.usu.edu

This work was supported by Grant from the National Institute for Environmental Health Sciences, ES05814


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Although the mechanism by which asbestos causes cancer remains unknown, iron associated with asbestos is thought to play a role in the pathogenic effects of fibers. Here, we examined the effects of asbestos on the epidermal growth factor receptor (EGFR) in human lung epithelial (A549) cells, human pleural mesothelial (MET5A) cells, and normal human small airway epithelial (SAEC) cells. Treatment of A549, MET5A, and SAEC cells with asbestos caused a significant reduction of EGFR tyrosine phosphorylation. This was both time- (15 min to 24 h) and concentration-dependent (1.5, 3, and 6 µg/cm2) in A549 cells. Also, treatment with 6 µg/cm2 crocidolite for 24 h diminished the phosphorylation levels of human EGFR 2 (HER2). Exposure of A549 cells to 6 µg/cm2 crocidolite for 3–24 h resulted in no detectable Y1045 phosphorylation and no apparent degradation of the EGFR. Inhibition of fiber endocytosis resulted in a considerable inhibition of EGFR dephosphorylation. Removal of iron from asbestos by desferrioxamine B or phytic acid inhibited asbestos-induced decreases in EGFR phosphorylation. The effects of crocidolite, amosite, and chrysotile on the EGFR phosphorylation state appeared to be directly related to the amount of iron mobilized from these fibers. These results strongly suggest that iron plays an important role in asbestos-induced inactivation of EGFR.

Key Words: asbestos • iron • human lung epithelial cells


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Asbestos is a naturally occurring mineral that has been used for centuries because of its unique physical and chemical properties, including durability, tensile strength, and heat resistance. However, it was recognized to be a human carcinogen in the late 1950s. Exposure to asbestos increases the risk of mesothelioma of the pleura and bronchogenic carcinoma of the lungs. Even though the molecular mechanism by which asbestos exerts these effects is not fully understood, the carcinogenicity of asbestos has been related to some of its physical properties, such as size and durability of fibers (1). The most carcinogenic forms of asbestos contain as much as 27% iron by weight. It has been shown that iron from asbestos catalyzes the generation of reactive oxygen species (ROS), such as superoxide radical (O2·–) and hydroxyl radical (·OH). In addition, iron has been shown to be responsible for O2 consumption, induction of DNA single-strand breaks, lipid peroxidation, and potentially cancer. There is also significant evidence suggesting that iron from other sources besides asbestos may cause human cancer.

Although the mechanism by which asbestos exerts its pathologic effects is not known, at least two plausible mechanisms should be considered. Asbestos-induced pulmonary toxicity might be initiated by ROS generated from mobilized iron via Haber-Weiss reactions (1), and possibly by interaction of asbestos fibers with the plasma membrane through a receptor-like mechanism (2).

Epidermal growth factor receptor (EGFR/ErbB1) is a transmembrane protein with intrinsic tyrosine kinase activity that is stimulated upon ligand binding. EGFR activation involves homo- and heterodimerization with other members of ErbB receptor family, such as ErbB2 (HER2), transphosphorylation of receptors, recruitment of various signaling proteins, and subsequently activation of many different downstream signaling pathways. It has been shown that a variety of stresses, including both chemical and physical agents, can activate EGFR. These include ultraviolet rays (3), hydrogen peroxide (H2O2) (4), and asbestos fibers (2, 5–8). Inactivation of the EGFR can lead to G1 arrest (9) and apoptosis (10). Both activation and inactivation of the EGFR are associated with pathologic effects.

The majority of studies of interaction between asbestos and EGFR have been done in rodent cells or rodents, e.g., rat pleural mesothelial (RPM) cells (2, 6, 11), murine alveolar type II epithelial (C10) cells, and transgenic mice (8). The treatment of rodent cells led to increased expression, phosphorylation, and subsequent activation of the EGFR, leading to cell injury (apoptosis) and/or proliferation. Inhibition of the asbestos-induced phosphorylation of EGFR blocked the development of apoptosis in RPM cells. Studies with human cells are very limited and involve investigation of EGFR on the plasma membrane using only microscopy (5). In an immunohistochemical study of EGFR after treatment of human pleural mesothelial (MET5A) cells and human lung epithelial (A549) cells with long (>= 60 µm) asbestos fibers, MET5A cells showed patterns of aggregation and increases in EGFR protein, whereas no effects were observed in A549 cells. It was concluded that this might be due to some differences in sensitivity to cytotoxic effects of asbestos and/or different mechanisms of response between mesothelial cells and epithelial cells.

The purpose of this study was to investigate and compare the effects of asbestos fibers on the EGFR activity in relevant human lung and pleural target cells, e.g., epithelial (A549 and small airway epithelial cells [SAEC]) and mesothelial (MET5A) cells, and to determine if iron associated with fibers plays some role in this process. Our results indicated that in contrast to rodent cells, treatment of human lung epithelial and pleural mesothelial cells with asbestos fibers resulted in dephosphorylation and subsequent inactivation of the ErbB family of receptor tyrosine kinases. In addition, it appeared that iron was responsible, at least in part, and that the effect on the receptor was mediated by internalized fibers. We have identified a potentially important mechanism by which relevant human lung and pleural target cells respond to asbestos fibers.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
An A549 cell line (12), MET5A cell line (5), and primary cultures of human SAEC cells were used for these studies.

Methods for Western blotting and immunoprecipitation (13), in vitro kinase phosphorylation assays (6), and biotin labeling (4) have previously been described.

The role of iron was examined by treating the cells with an iron chelator, phytic acid, or with fibers from which the redox active iron had been removed with another iron chelator, desferrioxamine B (14).

To inhibit endocytosis of fibers, the cells were cultured in the presence of cytochalasin B or D.

The entire MATERIAL AND METHODS can be viewed in the online supplement.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Effects of EGF on EGFR Phosphorylation
To investigate if A549 cells, originally obtained from an alveolar carcinoma, respond to EGF as do normal cells, serum-deprived A549 cells were treated with EGF at 100 ng/ml. Consistent with previous findings by Goldkorn and coworkers (15), the addition of EGF stimulated a rapid autophosphorylation of the EGFR, as measured by Western blotting analysis using antibodies recognizing either the total EGFR or the EGFR phosphorylated on Y1068 and Y1173 (see Figure E1A in the online supplement). The tyrosine phosphorylation peaked between 15 and 30 min after the addition of EGF and then rapidly declined. We also investigated the effects of EGF on kinase activation of the EGFR by means of a traditional in vitro kinase assay using [{gamma}-32P] ATP and the universal protein tyrosine kinase substrate poly (Glu, Tyr, 4:1) (Figure E1B). Consistent with Western blotting results, EGF markedly increased the phosphorylation level of both the receptor (data not shown) and the substrate above that of the control, untreated cells. A 4.7-fold increase was observed with the peak response after 30 min. Taken together, these results demonstrated that A549 cells showed normal responses to EGF.

Effects of Three Types of Asbestos Fibers on EGFR Phosphorylation
We comparatively examined the ability of three types of asbestos fibers to modulate the phosphorylation state of EGFR. We observed that asbestos treatment led to a significant decrease in the phosphorylated (upper band) and an increase in the unphosphorylated (lower band) form of EGFR, respectively (Figure 1). In addition, in contrast to the positive control, all three types of asbestos fibers significantly decreased constitutive phosphorylation of Y1068 and Y1173. The amount of decrease in phosphorylation observed after crocidolite, amosite, or chrysotile treatment appeared to be directly related not only to the percentage of iron by weight present in the different forms, 27, 27, and 2%, respectively, but also to the amount of iron mobilized from these fibers (16).



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  		Figure 1. Effects of three types of asbestos fibers on EGFR phosphorylation. Serum-deprived A549 cells were treated with 6 µg/cm2 crocidolite (CRO), amosite (A), or chrysotile (short and intermediate range; CSR and CIR, respectively) for 24 h, or with 100 ng/ml EGF for 15 min (positive control). Cellular lysates were subjected to Western blotting analysis, as described in MATERIALS AND METHODS. Phosphorylation of Y1068 and Y1173 of the EGFR was detected using the indicated antibodies. EGFR levels were detected using an anti-EGFR antibody (the upper and lower bands represent phosphorylated and unphosphorylated EGFR forms, respectively). ß-Actin antibody was used to demonstrate equal protein loading. Data presented are representative of three separate experiments.

 
Because Zanella and colleagues (6) have shown that asbestos induces EGFR phosphorylation rather than dephosphorylation in RPM cells, we analyzed if different types of F12 media (Kaighn's modification, with or without Fe) or serum (dialyzed or normal FBS) could change the effects of asbestos on EGFR in A549 cells. We observed that neither medium nor serum had an impact on asbestos-induced EGFR dephosphorylation in A549 cells (data not shown).

Effects of Crocidolite on ErbB2/HER2 Phosphorylation
Because HER2, one of the four members of the ErbB receptor family, acts synergistically with EGFR and both are thought to interact with each other by transphosphorylation via heterodimer formation, we examined the effect of asbestos fibers on HER2 membrane receptors (Figure E2). Consistent with the results with EGFR, crocidolite treatment decreased the constitutive phosphorylation levels on Y877, the site involved in regulation of HER2 biological activity, and Y1248, one of the major autophosphorylation sites that couples HER2 to the Ras-Raf-MAP kinase signal transduction pathway. Interestingly, we also observed a minor, time-dependent decrease in the total level of HER2 protein, suggesting that HER2 inactivation could be coupled to its subsequent degradation. This indicates that crocidolite treatment is able to affect not only EGFR, but also another member of the ErbB family of receptor tyrosine kinases.

Effects of Crocidolite on EGFR Activation, Time- and Dose-Dependence
Because asbestos decreased the constitutive EGFR phosphorylation, it was necessary to determine whether this phenomenon was time- and/or concentration-dependent. For the remainder of the study, only crocidolite was used. EGFR dephosphorylation after crocidolite treatment occurred within the first 3 h with a peak response at 24 h (almost total disappearance of phosphorylation bands) (Figure 2A). Furthermore, we also investigated the ability of crocidolite to modulate the in vitro kinase activity of the EGFR (Figure 2B). Crocidolite treatment decreased the phosphorylation of the receptor (data not shown) and decreased the phosphorylation activity of EGFR on the substrate below that of the control, untreated cells.




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  		Figure 2. Effects of crocidolite and TiO2 on EGFR activation. Time dependence. (A) Serum-deprived A549 cells were incubated with 6 µg/cm2 crocidolite (CRO) for the indicated time intervals, or with 100 ng/ml EGF for 15 min (positive control). Cellular lysates were subjected to Western blotting analysis, as described in MATERIALS AND METHODS. Phosphorylation of Y1068 and Y1173 of the EGFR was detected using the indicated antibodies. EGFR levels were detected using an anti-EGFR antibody (the upper and lower bands represent phosphorylated and unphosphorylated EGFR forms, respectively). (B) Protein aliquots (1 mg) from untreated or crocidolite-treated A549 cells were subjected to immunoprecipitation with anti-EGFR, and to traditional in vitro kinase assays with the substrate poly (Glu, Tyr, 4:1), as described in MATERIALS AND METHODS. (C) Serum-deprived A549 cells were incubated with 6 µg/cm2 crocidolite (CRO) or TiO2 for the indicated time intervals. Cellular lysates were subjected to Western blotting analysis, as described above. ß-Actin antibody was used to demonstrate equal protein loading. The in vitro kinase assay and Western blotting experiments were repeated two and three times, respectively, and representative autoradiograms are shown. The optical densities of the bands were quantified. The results are shown as the mean ± SE, and are expressed as fold increase relative to the untreated control. Error bars represent the SE. *P < 0.05 compared with control, untreated groups.

 
Others have observed that TiO2 particles do not cause cell apoptosis (17) or a change in mitochondrial membrane potential ({Delta}{psi}m) (18, 19). Thus, we also investigated the effects of TiO2 on the EGFR activity. In contrast to crocidolite, TiO2 had no effect on the EGFR phosphorylation state (Figure 2C). This is consistent with previous findings that TiO2 did not affect the cells.

Treatment of cells with three different concentrations of crocidolite resulted in a dose-dependent decrease in the EGFR phosphorylation (Figure 3A). A strong association between crocidolite concentration and the receptor dephosphorylation was observed.




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  		Figure 3. Effects of crocidolite on EGFR activation. Dose dependence. (A) Serum-deprived A549 cells were incubated with three different concentrations of crocidolite (CRO) (1.5, 3, or 6 µg/cm2) for 24 h. Cellular lysates were subjected to Western blotting analysis, as described in MATERIALS AND METHODS. Phosphorylation of Y1068 and Y1173 of the EGFR was detected using the indicated antibodies. EGFR levels were detected using an anti-EGFR antibody (the upper and lower bands represent phosphorylated and unphosphorylated EGFR forms, respectively). (B) MET5A cells were incubated with or without crocidolite (1.5, 3, or 6 µg/cm2) for 24 h, and cellular lysates were subjected to Western blotting analysis, as described above. (C) SAEC cells were incubated with or without crocidolite (3 and 6 µg/cm2) for 24 h, and cellular lysates were subjected to Western blotting analysis, as described above. (D) Protein aliquots (1 mg) from untreated or crocidolite-treated A549 cells were subjected to immunoprecipitation with anti-EGFR, and to traditional in vitro kinase assays with the substrate poly (Glu, Tyr, 4:1), as described in MATERIALS AND METHODS. ß-Actin antibody was used to demonstrate equal protein loading. The in vitro kinase assay and Western blotting experiments were repeated two and three times, respectively, and representative autoradiograms are shown. The optical densities of the bands were quantified. The results are shown as the mean ± SE, and are expressed as fold increase relative to the untreated control. Error bars represent the SE. *P < 0.05 compared with control, untreated groups.

 
Importantly, MET5A and normal human small airway epithelial cells showed similar responses to crocidolite, suggesting that the responses observed in A549 cells were representative of transformed and normal human cells (Figures 3B and 3C).

Next, we investigated if exposure of A549 cells to three concentrations of crocidolite for 24 h could affect the in vitro kinase activity of the EGFR (Figure 3D). In comparison to untreated control cells exhibiting constitutive levels of phosphorylation, crocidolite treatment (1.5, 3, or 6 µg/cm2) decreased the phosphorylation activity of EGFR on the substrate below that of the control by ~ 2- to 4-fold in a dose-dependent manner.

Effects of Crocidolite on EGFR Degradation
Because lysosomal targeting of EGFR has been shown to be independent of the tyrosine kinase activity (20), we investigated if asbestos-induced EGFR dephosphorylation was coupled to receptor degradation. Phosphorylation of Y1045 in the EGFR creates a major docking site for c-Cbl, which in turn enables receptor ubiquitination and degradation. Thus, we analyzed the phosphorylation status of Y1045 and the levels of EGFR before and after asbestos treatment. In addition, because Zhuang and coworkers (21) showed that oxidative stress led to a caspase-mediated EGFR degradation during the early phase of cell death, we followed the fate of the surface EGFR more specifically with Biotin-X-NHS. Consistent with observations by Ravid and colleagues (4), 15 min EGF treatment resulted in significant Y1045 phosphorylation (Figure 4A). Also, in the presence of 100 ng/ml EGF for 9 h, the levels of biotinylated EGFR were dramatically decreased (Figure 4B). In contrast, crocidolite exposure from 3–24 h resulted in negligible Y1045 phosphorylation with no apparent decrease in biotinylated EGFR (Figures 4A and 4B). Thus, crocidolite treatment did not lead to EGFR degradation.



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  		Figure 4. Effects of crocidolite on EGFR degradation. (A) Serum-deprived A549 cells were incubated with or without 6 µg/cm2 crocidolite (CRO) for the indicated time intervals, or with 100 ng/ml EGF for 15 min (positive control). Cellular lysates were subjected to Western blotting analysis, as described in MATERIALS AND METHODS. Phosphorylation of Y1045 of the EGFR was detected using the indicated antibody. ß-Actin antibody was used to demonstrate equal protein loading. (B) Cell surface proteins of serum-deprived A549 cells were biotinylated, followed by incubation with 6 µg/cm2 crocidolite for the indicated time intervals, or with 100 ng/ml EGF for 9 h (positive control). Biotin protein labeling was detected with horseradish peroxidase–conjugated streptavidin, as described in MATERIALS AND METHODS. The experiments were repeated three times, and representative autoradiograms are shown.

 
Effects of Cytochalasins, Desferrioxamine B, and Phytic Acid on Crocidolite-Induced EGFR Inactivation
We have previously observed that cytochalasins, which block actin polymerization, could be successfully used in A549 cells as inhibitors of endocytosis (22, 23). To determine whether asbestos-induced EGFR inactivation required the uptake of fibers, before and during treatment, cells were cultured in the presence of cytochalasin B or D (CB or CD). As shown in Figure 5A, CB and CD, alone, had negligible effect on EGFR phosphorylation. However, treatment of A549 cells with crocidolite in the presence of CB or CD (CB/CRO or CD/CRO) resulted in a considerable inhibition of EGFR dephosphorylation. Notably, cytochalasin D treatment resulted in a much higher inhibition of receptor dephosphorylation than respective treatment with CB. Nevertheless, co-treatment of A549 cells with fibers and either cytochalasin did not result in a full recovery of receptor phosphorylation levels. Taken together, the results presented here strongly suggest that fiber endocytosis is an important step for EGFR dephosphorylation.




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  		Figure 5. Effects of cytochalasins, desferrioxamine B, and phytic acid on crocidolite-induced EGFR inactivation. (A) Serum-deprived A549 cells were pretreated with or without 5 µg/ml cytochalasin D or B (CD and CB) for 1 h, followed by incubation with 6 µg/cm2 crocidolite for 24 h (CRO). Cellular lysates were subjected to Western blotting analysis, as described in MATERIALS AND METHODS. Phosphorylation of Y1068 and Y1173 of the EGFR was detected using phospho-specific antibodies. EGFR levels were detected using an anti-EGFR antibody (the upper and lower bands represent phosphorylated and unphosphorylated EGFR forms, respectively). (B) Serum-deprived A549 cells were incubated with three different concentrations of crocidolite (CRO), 1.5, 3, or 6 µg/cm2, for 24 h, or fibers from which the redox active iron had been removed with desferrioxamine B (DF-CRO). Cellular lysates were subjected to Western blotting analysis, as described above. (C) 1 mg protein aliquots from crocidolite- or DF-CRO–treated A549 cells were subjected to immunoprecipitation with anti-EGFR, and to traditional in vitro kinase assays with the substrate poly (Glu, Tyr, 4:1), as described in MATERIALS AND METHODS. (D) Serum-deprived A549 cells were treated with 6 µg/cm2 DF-CRO for 24 h, or the cells were pretreated with or without 500 µM phytic acid (PA) for 1 h, followed by incubation with 6 µg/cm2 crocidolite for 24 h (PA/CRO). Cellular lysates were subjected to Western blotting analysis, as described above. ß-Actin antibody was used to demonstrate equal protein loading. The in vitro kinase assay and Western blotting experiments were repeated two and three times, respectively, and representative autoradiograms are shown. The optical densities of the bands were quantified. The results are shown as the mean ± SE, and are expressed as fold increase relative to the untreated control. Error bars represent the SE. *P < 0.05 compared with 6 µg/cm2 crocidolite.

 
We have previously shown that the pretreatment of asbestos fibers (24) and coal fly ash (25) with an iron chelator desferrioxamine B (DF-crocidolite and -CFA, respectively) greatly reduced the ability of these particulates to exert their pathologic effects. To determine if iron associated with crocidolite was involved in EGFR inactivation, serum-deprived A549 cells were treated with three concentrations of DF-crocidolite (1.5, 3, or 6 µg/cm2) for 24 h. Iron removal from crocidolite was able to greatly inhibit the amount of the EGFR dephosphorylation, compared with that seen with fibers not treated with a chelator (Figure 5B). Furthermore, we also investigated the ability of DF-crocidolite to modulate the in vitro kinase activity of the EGFR (Figure 5C). Consistent with the Western blotting analysis, incubation of cells with DF-crocidolite radically inhibited dephosphorylation levels of the receptor (data not shown) and inhibited EGFR phosphorylation activity on the substrate compared with that seen with fibers not treated with a chelator. Further evidence implying an important role for iron in the EGFR inactivation was that fibers incubated with phytic acid (PA), another iron chelator, also decreased the levels of receptor dephosphorylation (Figure 5D). PA alone had no effect on the EGFR phosphorylation level. Noteworthy is the fact that DF-pretreated fibers inhibited crocidolite-induced EGFR inactivation to a much greater extent than crocidolite co-incubated with PA. Taken together, the correlation between iron removal from crocidolite fibers and inhibition of receptor dephosphorylation clearly supports the involvement of iron in the inactivation of EGFR.


   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
In the present study we observed that asbestos treatment of human lung epithelial and pleural mesothelial cells resulted in EGFR dephosphorylation and inactivation. This phenomenon may represent a potentially important mechanism by which relevant human lung and pleural target cells respond to asbestos fibers. The results presented here strongly suggest that the fibers must be internalized for the EGFR to be inactivated, and that the iron associated with fibers was directly or indirectly responsible for some of this effect. We also found that asbestos fibers modulate not only EGFR, but also another member of the ErbB family of receptor tyrosine kinases (i.e., HER2). Understanding the role of EGFR inactivation in asbestos-induced multiple effects on target cells may provide new insights into the process of carcinogenesis.

The interaction between asbestos and EGFR has mostly been investigated in rodent cells and rodents. Two research groups have observed that asbestos treatment of RPM cells induced EGFR phosphorylation (2, 6, 11). Increases in EGFR mRNA and protein synthesis have also been noted (2). In addition, Manning and colleagues (8) have observed EGFR-mediated proto-oncogene expression and cell proliferation in transgenic mice and murine C10 cells. The results presented here support other studies in the literature showing differences between rodent and human tissue in terms of resistance to genotoxicity and/or cytotoxicity by asbestos (26).

On the other hand, the interaction between asbestos and EGFR has not been thoroughly explored in humans. The studies were limited to microscopic work, as reported by Pache and coworkers (5). Interestingly, they observed that MET5A cells showed patterns of aggregation and increases in EGFR protein on the surface of cells phagocytizing long asbestos fibers (>= 60 µm), whereas no changes were observed in A549 cells. Our observations reported herein are inconsistent with that work. A comparative examination of human A549 and MET5A cells showed that patterns of EGFR dephosphorylation caused by asbestos were similar. In both cell lines the amount of decrease in phosphorylation correlated with dose and time.

A549 cells are one of the cultured human lung epithelial cell lines being used as a tissue culture model of lung epithelial cells in research. However, because A549 cells were originally obtained from an alveolar carcinoma, there is concern that they may not be a faithful representation of normal cells. However, in the studies presented here, A549 cells responded in the same way to crocidolite as normal human SAEC. We have previously shown that A549 cells responded similarly to particles (glutathione efflux, ferritin induction) when compared with SAEC and that the responses were not an artifact of a single cell type (27). This was complemented by our findings that crocidolite treatment of both SAEC and A549 cells resulted in a decrease in the EGFR phosphorylation levels in a dose-dependent manner. We conclude that the responses observed in A549 cells are representative of not only mesothelial and epithelial cells, but also of normal and transformed human lung cells.

We have also observed that in contrast to EGF-treated cells, EGFR remains at the plasma membrane throughout the 24-h exposure time to crocidolite, suggesting that receptor dephosphorylation is not coupled to its degradation. Because there are several pathways leading to receptor degradation, e.g., lysosome or proteasome, we used two distinctive approaches to investigate this. We observed that asbestos failed to initiate not only ubiquitin-mediated lysosomal targeting but also alternative mechanisms of receptor downregulation. Interestingly, crocidolite treatment led to a minor decrease in the total level of HER2 protein, suggesting that HER2 dephosphorylation could be coupled to its subsequent degradation.

Inert particulates, such as TiO2 or glass beads, do not cause any pathologic effects in either mesothelial (28, 29) or epithelial cells (17–19). Consistent with that, we observed that significant decreases in the EGFR phosphorylation levels occurred with crocidolite, but not with TiO2. The fact that iron on the asbestos fibers appears to be related to EGFR dephosphorylation may explain the differences observed between TiO2 and asbestos.

There is significant evidence demonstrating that iron was mobilized intracellularly from asbestos fibers and was responsible for O2 consumption, lipid peroxidation, and DNA oxidation. It has been proposed that iron mobilized from asbestos into a low-molecular-weight fraction led to pathologic phenomena, including cancer. DF and PA, which bind tightly to all of the iron coordination sites making it inert, can prevent iron-derived reactive oxygen species production within cells. We found that iron chelators had inhibitory effects on crocidolite-induced EGFR dephosphorylation. Noteworthy is the fact that the long-term removal of bioavailable iron from crocidolite by DF before exposure to cultured cells inhibited EGFR inactivation to a much greater extent than fibers co-incubated with PA. We also observed that the amount of EGFR dephosphorylation in A549 cells by two types of asbestos, crocidolite and amosite, was significantly greater than that by the third type, chrysotile, either short or intermediate-length fibers. Also, direct comparison of short and intermediate length chrysotile fibers revealed that intermediate fibers seemed to be somewhat more toxic to A549 cells than short fibers, which could be due to differences in the fiber dimensions and/or iron bioavailability. Most importantly, the observed effects of crocidolite, amosite and chrysotile on the EGFR phosphorylation state appeared to be related to the amount of iron mobilized from these fibers (16). Thus, there appeared to be a correlation between iron mobilization from asbestos and the degree of EGFR dephosphorylation.

Because the EGFR is an integral membrane protein with crucial domains on both sides of the plasma membrane, we examined whether the process of fiber endocytosis was necessary for its inactivation. We observed that the endocytosis inhibitor cytochalasin considerably inhibited EGFR dephosphorylation. Interestingly, analysis of the results revealed that CD treatment resulted in a much greater inhibition of receptor dephosphorylation caused by crocidolite than respective treatment with CB. This phenomenon could be simply explained by the fact that CD is a much more potent inhibitor of actin polymerization than CB. Endocytosis is also important for other asbestos-mediated effects in lung epithelial cells (12) and mesothelial cells (30, 31). We have previously observed that endocytosis of crocidolite in A549 cells was required for iron mobilization (12). This uncontrolled entry of iron may represent "iron overload" in cells endocytizing the fibers, which could be responsible for many asbestos-dependent biological effects. These observations, taken together, suggest that the effects of asbestos on EGFR are mediated intracellularly, not extracellularly.

It is apparent that iron associated with crocidolite was required, at least in part, for EGFR dephosphorylation. The primary mechanism by which iron from asbestos fibers leads to receptor inactivation is not yet clear. Iron can act as both an oxidant and a reductant, directly or indirectly through generation of ROS. Kamata and coworkers (32) have shown that redox-sensitive cellular processes could inactivate EGFR. Direct treatment of cells with H2O2 activates, rather than inactivates, EGFR activity (4). These treatments could have had extracellular, as well as intracellular effects and certainly would expose cells to much higher levels of H2O2 then would ever be generated by asbestos. Also, as discussed earlier, it appears that asbestos exerts its effects intracellularly (12). It is possible that asbestos may be activating protein phosphatases that dephosphorylate and inactivate the EGFR. Zhuang and colleagues (21) have recently observed that exposure of human keratinocytes to singlet oxygen resulted in rapid decreases in EGFR phosphorylation through activation of protein phosphatases.

Activation of EGFR and its signaling is believed to be an important contributor to the growth and survival of cells in a variety of conditions. Thus, the dephosphorylation and inactivation of EGFR observed after asbestos treatment may be one step in the pathway leading to apoptosis. Stimuli other than asbestos have also been shown to inhibit EGFR phosphorylation, resulting in G1 arrest, upregulation of p27 (9), and subsequent apoptosis (10). Recent evidence strongly suggests a relationship between inactivation of EGFR-associated survival pathways and stimulation of the stress-activated protein kinases in terms of a balance between cell death and proliferation. The emerging role of EGFR inactivation in relevant human lung and pleural target cells may be of critical importance in the process of apoptosis. Several groups have independently provided strong evidence that asbestos triggers apoptosis in mesothelial (2, 28, 29, 33–35) and epithelial cells (36), including A549 cells (17–19). Apoptosis represents an important mechanism critical for elimination of defective cells without stimulating an inflammatory response. However, extensive cell death due to apoptosis can lead to compensatory proliferation of surrounding cells. If these replicating cells have already sustained critical mutations, then replication could promote formation of a tumor. We are currently investigating the signaling events responsible for asbestos-induced EGFR dephosphorylation and apoptosis in human lung epithelial cells.

In conclusion, we observed that crocidolite treatment of A549, MET5A, and SAEC cells resulted in EGFR dephosphorylation and inactivation. This effect appeared to be mediated, at least in part, by the presence of bioavailable iron, and appeared to be mediated intracellularly. Inactivation of EGFR may be an important step in the pathway leading to apoptosis. Elucidation of molecular mechanisms underlying asbestos-induced DNA damage and apoptosis may lead to a better understanding of asbestos-related pulmonary toxicity.


   Acknowledgments
 
The authors thank Dr. Sun-Hee Park, P. Pande, T. Musleh, and L. Buelow for their support and helpful discussions. They are grateful to Jed Benson, Gregg Marshall, and Ren Gonzales for excellent technical assistance.


   Footnotes
 
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Conflict of Interest Statement: A.B. has no declared conflicts of interest; and A.E.A has no declared conflicts of interest.

Received in original form April 26, 2004

Received in final form December 22, 2004


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

  1. Hardy JA, Aust AE. Iron in asbestos chemistry and carcinogenicity. Chem Rev 1995;95:97–118.
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