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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 51-60, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0013OC

Asbestos Inhalation Induces Tyrosine Nitration Associated with Extracellular Signal-Regulated Kinase 1/2 Activation in the Rat Lung

Akitaka Iwagaki, Nonghoon Choe, Yingyue Li, David R. Hemenway and Elliott Kagan

Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, Maryland; and Department of Civil and Environmental Engineering, University of Vermont, Burlington, Vermont

Address correspondence to: Elliott Kagan, M.D., Department of Pathology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799. E-mail: ekagan{at}usuhs.mil


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nitration of proteins by peroxynitrite (ONOO-) has been shown to critically alter protein function in vitro. We have shown previously that asbestos inhalation induced nitrotyrosine formation, a marker of ONOO- production, in the rat lung. To determine whether asbestos-induced protein nitration may affect mitogen-activated protein kinase (MAPK) signaling pathways, lung lysates from crocidolite and chrysotile asbestos-exposed rats and from sham-exposed rats were immunoprecipitated with anti-nitrotyrosine antibody, and captured proteins were subjected to Western blotting with anti–phospho-extracellular signal-regulated kinase (ERK)1/2 antibodies. Both types of asbestos inhalation induced significantly greater phosphorylation of ERK1/2 in rat lung lysates than was noted after sham exposure. Phosphorylated ERK proteins co-immunoprecipitated with nitrotyrosine. Moreover, in MAPK functional assays using Elk-1 substrate, immunoprecipitated phospho-ERK1/2 in lung lysates from both crocidolite-exposed and chrysotile-exposed rats demonstrated significantly greater phosphorylation of Elk-1 than was noted after sham exposure. Asbestos inhalation also induced ERK phosphorylation in bronchoalveolar lavage cells. Lung sections from rats exposed to crocidolite or chrysotile (but not from sham-exposed rats nor from rats exposed to "inert" carbonyl iron particles) demonstrated strong immunoreactivity for nitrotyrosine and phospho-ERK1/2 in alveolar macrophages and bronchiolar epithelium. These findings suggest that asbestos fibers may activate the ERK signaling pathway by generating ONOO- or other nitrating species that induce tyrosine nitration and phosphorylation of critical signaling molecules.

Abbreviations: bovine serum albumin, BSA • bronchoalveolar lavage, BAL • epidermal growth factor receptor, EGFR • extracellular signal-related kinase, ERK • fetal bovine serum, FBS • inducible form of nitric oxide synthase, iNOS • mitogen-activated protein kinase, MAPK • mitogen-activated protein kinase kinase/ERK kinase, MEK-1 • nitric oxide, •NO • nuclear factor-{kappa}B, NF-{kappa}B • peroxynitrite, ONOO- • phosphate-buffered saline, PBS • protein kinase C, PKC • rat pleural mesothelial cells, RPMC • sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE • superoxide dismutase, SOD • Tris-buffered saline Tween-20, TBST


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Inhalation of asbestos fibers can induce a variety of fibrotic and malignant diseases, including visceral pleural fibrosis, parietal pleural plaques, asbestosis, bronchogenic carcinoma, and diffuse malignant mesothelioma of the pleura and peritoneum (1, 2). Although epidemiologic studies have established that all commercial types of asbestos can induce pleural and pulmonary injury in occupationally exposed individuals (3, 4), there has been considerable debate regarding the differing potential of chrysotile (a serpentine form of asbestos that was used widely in industry in North America) versus amphibole asbestos to inflict clinically detectable injury (5, 6). There is evidence that asbestos fibers can stimulate gene expression in diverse types of cells via several intracellular transduction pathways, including those involving the protein kinase C (PKC), nuclear factor (NF)-{kappa}B, p38 kinase, and extracellular signal-regulated kinase (ERK) signaling cascades (7–11). Furthermore, iron-rich crocidolite asbestos fibers have been shown to generate hydroxyl radical (•OH) formation via superoxide (•O2-)-driven, iron-catalyzed Haber-Weiss (Fenton) reactions, and these reactions have been implicated in asbestos-induced injury (12, 13). Because oxidant stress has been demonstrated to activate several different signaling pathways (14, 15), it has been speculated that Fenton-type reactions may be responsible for mediating the oncogenic and fibrogenic effects of asbestos, by triggering activation of diverse cell signaling cascades (16).

Recent studies have indicated that asbestos fibers can stimulate the formation of reactive nitrogen species in lung and pleural cells. Both chrysotile and crocidolite exposure have been shown to upregulate expression of the inducible form of nitric oxide synthase (iNOS) and to stimulate nitric oxide (•NO) formation in rat pleural mesothelial cells and in rat alveolar and pleural macrophages (17–19). Moreover, when inhaled, both commercial types of asbestos induced the formation of nitrotyrosine, a surrogate marker of peroxynitrite anion (ONOO-) production, in the lungs and pleura of the rat (19). However, the latter study did not identify the precise protein targets undergoing protein nitration.

The present study was undertaken to determine whether asbestos inhalation can induce nitration of specific lung proteins, and to assess whether nitration of tyrosine residues may affect the functional activity of the targeted proteins. Using a defined model of asbestos-induced pulmonary injury, we show for the first time that both crocidolite and chrysotile asbestos fibers can induce in vivo nitration and protracted ERK functional activation in the rat lung 2 wk after the cessation of asbestos exposure. Furthermore, we have identified strong immunoreactivity for both nitrotyrosine and phospho-ERK1/2 in the airway epithelium and alveolar macrophages of crocidolite- as well as chrysotile-exposed rats, but not in sham-exposed rats nor in rats exposed to the "inert particulate" carbonyl iron.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mineral Dust Samples and Reagents
International Union Against Cancer (UICC) crocidolite fibers were a generous gift from Dr. David Rees (National Centre for Occupational Health, Johannesburg, South Africa). National Institute of Environmental Health Sciences (NIEHS, Research Triangle Park, NC) chrysotile fibers were obtained from the NIEHS. 3-Nitro-L-tyrosine, O-phospho-L-tyrosine, bovine serum albumin (BSA), sodium dithionite, sodium dodecyl sulfate (SDS), sodium deoxycholate, PMSF, aprotinin, antipain, leupeptin, pepstatin A, Na3VO4, NaCl, EDTA, NaF, Na2CO3, NaHCO3, KH2PO4, Na2P2O7, chymotrypsin, 3,3'-diaminobenzidine, and Harris' hematoxylin were purchased from Sigma Chemical Co. (St. Louis, MO). Peroxynitrite was obtained from Upstate Biotechnology (Lake Placid, NY). Tris base, Triton X-100, blocking reagent, and leupeptin were supplied by Boehringer Mannheim (Indianapolis, IN). Diff-Quik Stain Set was obtained from Baxter Healthcare (McGaw Park, IL), and sodium pentobarbital was purchased from Abbott Laboratories (North Chicago, IL). RPMI 1640, fetal bovine serum, and phosphate-buffered saline (PBS) were supplied by Biofluids, Inc. (Rockville, MD). Protein A–bound beads were purchased from Amersham (Arlington Heights, IL).

Animals and Inhalation Exposure Regimen
For inhalation exposures, three groups of adult male Fischer-344 rats were placed in whole body inhalation chambers and exposed to either UICC crocidolite asbestos, NIEHS chrysotile asbestos, or filtered room air (sham-exposed group). Asbestos aerosols were generated via a modified Timbrell dust generator (BGI, Waltham, MA). Total fiber mass aerosol concentrations were measured by standard gravimetric analysis on 0.8 µM pore membrane filters, as described previously (19). The time-weighted mean asbestos exposure concentrations (mean ± SE) were 15.17 ± 7.78 mg/M3 for crocidolite and 8.52 ± 0.81 mg/M3 for chrysotile. These exposure levels were comparable with historic asbestos dust concentrations recorded in the workplace environment of asbestos mines and mills (20). The aerosols were highly respirable. An established inhalation exposure protocol was used, in which the three groups of rats were exposed for 6 h/d on 5 d/wk over 2 wk (12, 19). All the rats were killed 14 d after the cessation of exposure by the intraperitoneal administration of sodium pentobarbital (50 mg/kg), followed by exsanguination via the abdominal aorta.

Collection of Bronchoalveolar Lavage Cells
The technique of bronchoalveolar lavage (BAL) was performed as described previously (19). Briefly, after inserting a tube into the trachea, the lungs were lavaged six times with 7 ml of PBS and prewarmed to 37°C as described elsewhere. Because only a limited amount of protein was extractable from BAL cells, these cells were used exclusively in Western blot studies for the detection of phosphorylated ERK proteins, but not for immunoprecipitation experiments or for functional ERK kinase assays. Lavage cell cytospin preparations (2.5 x 104 cells/sample) were obtained using a Cytospin 3 cytocentrifuge (Shandon, Pittsburgh, PA), and were examined for immunocytochemical studies of phosphorylated ERK1/2 protein expression.

Western Blot Analyses
For Western blot analyses, the lungs were aseptically removed and freshly prepared tissues were frozen in liquid nitrogen. Lung tissue samples smashed on dry ice and BAL cells were solubilized in ice-cold cell lysing buffer, comprising 10 mM Tris-HCl (pH 7.6), 5 mM EDTA, 50 mM NaCl, 30 mM Na2P2O7, 50 mM NaF, 1 mM Na3VO4, 1% Triton X-100, 1 mM PMSF, 1 g/ml aprotinin, 1 g/ml antipain, 1 g/ml leupeptin, 1 g/ml pepstatin, and 1 g/ml chymostatin. After sonication for 30 s on ice, the lysates were centrifuged for 4 h at 4°C, after which the samples were solubilized in ice-cold lysing buffer and clarified by centrifugation at 25,000 x g for 30 min at 4°C. The supernatants were collected and their protein concentrations were determined, for normalization of protein levels, using a Bio-Rad Protein Reagent (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein were mixed 1:1 with 2x sample buffer, comprising 20% glycerol, 4.6% SDS, 10% ß-mercaptoethanol, 0.004% bromophenol blue, and 125 mM Tris-HCl (pH 6.8). The samples then were boiled and clarified by centrifugation. Subsequently, samples were loaded onto a 12% SDS-polyacrylamide gel electrophoresis (PAGE) gel, and run at 20 mA for 2 h. Proteins then were transferred wet to Immobilon-P Transfer Membranes (Millipore Corp., Bedford, MA), and stained with Coomassie blue, to verify equivalent transfer of samples. Membranes were blocked for 2 h at ambient temperature with Tris-buffered saline, comprising 50 mM Tris-HCl (pH 7.6) and 150 mM NaCl, and containing 1% BSA. Thereafter, the membranes were incubated overnight at 4°C with either mouse monoclonal anti-pan ERK (Upstate Biotechnology, Lake Placid, NY), mouse monoclonal anti–phospho-ERK1/ERK2 (New England Biolabs, Inc., Beverly, MA), or mouse monoclonal anti-nitrotyrosine (Cayman Chemical Co., Ann Arbor, MI). The primary antibodies were used at dilutions of 1:500 to 1:1,000. The blots were washed four times (10 min on each occasion) with Tris-buffered saline Tween-20 (TBST), comprising 50 mM Tris-HCl (pH 7.6), 150 mM NaCl and 0.1% Triton X-100, and incubated subsequently for 2 h at ambient temperature in Tris-buffered saline with horseradish peroxidase–conjugated anti-mouse IgG antibody (1:2,000 dilution; Sigma). Immunoreactive bands were visualized using the enhanced chemiluminescence (ECL) system (Amersham, Arlington Heights, IL) and quantified by densitometric analysis, using NIH image software.

For immunoprecipitation, equal amounts (500 µg) of lung protein lysates were incubated for 10 min at 4°C with protein A–bound beads, to eliminate nonspecific binding of protein. The lysates were clarified by centrifugation, whereupon the supernatants were incubated with 2 µg of monoclonal anti-pan ERK antibody or monoclonal anti-nitrotyrosine antibody for 2 h at 4°C. Antibody–antigen complexes were captured on protein A–bound beads during a 2-h incubation at 4°C. The beads were washed three times with lysing buffer and resolved in 2x sample buffer, boiled, and then clarified by centrifugation. Captured proteins then were subjected to Western blotting, as described above.

Elk-1 Kinase Functional Assays
To assess ERK1/ERK2 kinase functional activities, a commercially available nonradioactive kinase assay system was used (New England Biolabs). Accordingly, total lung proteins were extracted from lung lysates obtained from each exposure group of rats, as described above. One milligram of protein lysates were first incubated for 10 min at 4°C with protein A–bound beads (Amersham), to remove nonspecific binding of protein with the beads. Lysates then were clarified by centrifugation, and the supernatants were incubated with mouse monoclonal anti–phospho-ERK1/2 antibody (1:100 dilution; New England Biolabs) for 6 h at 4°C in the presence of protein A–bound beads. Antibody–antigen complexes were captured on protein A–bound beads during incubation. The beads were rinsed twice with 500 µl of cell lysing buffer and washed twice with 500 µl of 1x kinase buffer, comprising 25 mM Tris-HCl (pH 7.5), 5 mM ß-glycerolphosphate, 2 mM DTT, 0.1 mM Na3VO4, and 10 mM MgCl). The beads were incubated in 50 µl 1x kinase buffer with 2 µg of Elk-1 fusion protein in the presence of 200 µM ATP at 30°C for 30 min. The reaction was terminated with 50 µl of 2x sample buffer (20% glycerol, 4.6% SDS, 10% ß-mercaptoethanol, 0.004% bromophenol blue, and 125 mM Tris-HCl, pH 6.8). Thereafter, the samples were loaded onto a 12% SDS-PAGE gel and subjected to Western blot analysis with a rabbit polyclonal IgG anti–phospho-Elk-1 antibody (1:1,000 dilution; New England Biolabs), as described above. Purified ERK2 kinase (New England Biolabs) was used as a positive control. Immunoreactive bands were visualized using the ECL system. ERK-induced phosphorylation of Elk-1 fusion protein was quantified by densitometric analysis, using NIH image software.

Archival Rat Lung Specimens for Immunohistochemical Studies
Besides the lung specimens obtained from the three groups of exposed rats in this study, we were able to obtain additional archival rat lung specimens for immunohistochemical studies. The archival material, which was generously provided by Dr. Jing-Yao Liu and Dr. Arnold Brody from the Department of Pathology at Tulane University School of Medicine, comprised formalin-fixed, paraffin-embedded lung samples that were prepared originally for an unrelated, published study of transforming growth factor-{alpha} expression in asbestos-induced lung injury (21). The archival samples enabled us to compare the effects of both short-term (5-h) and long-term (2-wk) asbestos inhalation on nitrotyrosine formation and ERK phosphorylation in lung cells, and to determine whether any observable effects were unique for asbestos exposure. Archival specimens were obtained from three groups of rats (three rats/group) exposed by short-term (5-h) inhalation to either chrysotile asbestos, carbonyl iron particles, or room air (sham-exposed). Details of the exposure regimens were reported previously by Liu and coworkers (21). Carbonyl iron particles are nonfibrogenic and noncarcinogenic, and have been used as an "inert particulate" control both for in vitro and for in vivo studies of the biologic effects of asbestos fibers (18, 21).

Rat Pleural Mesothelial Cell Cultures
Rat pleural mesothelial cells (RPMC) were obtained and maintained in primary culture, as described previously (18). The cells were employed exclusively for experiments that were designed to test the specificity of the monoclonal anti-nitrotyrosine antibody in immunohistochemical studies. Before use, RPMC were fixed with 4% paraformaldehyde in PBS for 25 min.

Immunohistochemical Staining for Phospho-ERK1/2 and Nitrotyrosine
Immunohistochemical staining was performed on formalin-fixed rat lung sections from both current and archival samples. After deparaffinization, tissue sections were heated at 95°C for 10 min, to attain heat-induced epitope retrieval. The tissue sections then were incubated with 3% H2O2 in absolute methanol for 15 min at ambient temperature, to inhibit endogenous peroxidase activity. Specific immunohistochemical studies were performed for 2 h at ambient temperature, using mouse monoclonal anti–phospho-ERK1/2 antibody (10 µg/ml; New England Biolabs) and mouse monoclonal anti-nitrotyrosine antibody (10 µg/ml; Cayman Chemical) as primary antibodies. The tissue sections then were labeled, using a Universal DAKO LSBA 2 kit containing an appropriate, biotinylated secondary antibody and streptavidin-conjugated peroxidase (DAKO, Carpinteria, CA). For visualization, 3,3'-diaminobenzidine (Sigma) was used as a substrate, and the cell preparations and sections were counterstained with Harris' hematoxylin. As a negative control, the appropriate specific primary antibody was omitted, and 1% blocking reagent was substituted. As an additional negative control, tissue sections were incubated with normal mouse IgG2 in Tris buffer instead of the primary antibody.

As a positive control for nitrotyrosine immunostaining, cultured RPMC were treated with 200 µl of 24 mM ONOO- for 5 min, washed twice in PBS, incubated for 1 h with 400 µl of BSA in PBS at ambient temperature, then washed twice again in PBS before applying the primary anti-nitrotyrosine antibody. To confirm the specificity of the anti-nitrotyrosine antibody, the antiserum was incubated with either 10 mM 3-nitro-L-tyrosine or 10 mM O-phospho-L-tyrosine in 0.05 M Tris-HCl buffer immediately before the addition of the primary antibody to the ONOO--treated RPMC. Furthermore, to exclude the possibility of false-positive immunostaining for nitrotyrosine by the immunoperoxidase technique (22), ONOO-–treated RPMC were subsequently pretreated with 1 M sodium dithionite (which reduces nitrotyrosine to aminotyrosine) before adding the primary anti-nitrotyrosine antibody to the RPMC, as described previously (23).

Statistics
To determine whether there were any differences between exposure groups, data were analyzed by ANOVA using Bartlett's test. Individual comparisons between different exposure groups were made using Student's unpaired t test. Values of P < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Evidence of Protein Nitration in Rat Lungs
Western blot analyses for nitrated proteins in rat lung lysates immunoprecipitated with anti-nitrotyrosine antibody showed several distinct protein bands that ranged in molecular size from {bsim} 35–50 kD. Although lung lysates from the three rat exposure groups showed no obvious differences in the staining intensity of the majority of the nitrated protein bands, there were two distinct protein bands that were readily detectable in lung lysates from both crocidolite-exposed and chrysotile-exposed rats, but were not evident in lysates from sham-exposed rats (Figure 1). The molecular sizes of these nitrated proteins were 44 and 42 kD, respectively. Because the size range of the latter two proteins was similar to that of ERK1/2, additional studies were undertaken to determine whether the immunoprecipitated fractions contained ERK proteins.



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  		Figure 1. Immunoprecipitation of nitrated proteins in rat lung lysates from asbestos-exposed and sham-exposed rats. Lung lysate proteins were immunoprecipitated with anti-nitrotyrosine antibody, separated by SDS-PAGE, transferred to membranes, and then blotted with anti-nitrotyrosine antibody. Arrows indicate protein bands in the molecular size ranges of ERK1 and ERK 2 proteins, respectively. n = 3 rats per exposure group.

 
Asbestos Inhalation–Induced Phosphorylation of ERK Proteins in Rat Lung and BAL Samples
Western blot analyses showed that ERK1 and ERK2 proteins, as well as phospho-ERK1/2, were readily detected in the whole lung lysates (Figure 2) as well as BAL cell lysates (Figure 3) from the three groups of exposed rats. Whereas the total amounts of ERK proteins did not differ between the exposure groups in either whole lung or BAL lysate samples, there were discernable differences between sham-exposed and asbestos-exposed rats with respect to the amounts of phospho-ERK1/2 proteins recovered in the samples. Notably, significantly greater phosphorylation of ERK1/2 proteins was detected both in lung lysates (Figure 2) and in BAL cell lysates (Figure 3) after crocidolite or chrysotile exposure than after sham exposure. No statistically significant differences between the effects of chrysotile or crocidolite inhalation were noted in this regard.



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  		Figure 2. Western blots for total ERK1/2 and phosphorylated ERK1/2 proteins in lung lysates from asbestos-exposed and sham-exposed rats. Proteins were blotted with anti–pan-ERK (A) or anti–phospho-ERK1/2 antibodies (B) and quantified by densitometric analysis (C). n = 3 rats per exposure group. *P < 0.01 versus sham-exposed group.

 


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  		Figure 3. Western blots for total ERK1/2 and phosphorylated ERK1/2 proteins in BAL cell lysates from asbestos-exposed and sham-exposed rats. Proteins were blotted with anti–pan-ERK (A) or anti–phospho-ERK1/2 antibodies (B) and quantified by densitometric analysis (C). n = 3 rats per exposure group. *P < 0.05 versus sham-exposed group.

 
Asbestos Inhalation–Induced Upregulation of Elk-1 Phosphorylation by Rat Lung Immunoprecipitates
To determine whether the enhanced ERK phosphorylation that was detected in rat lung lysates from asbestos-exposed rats was accompanied by enhanced mitogen-activated protein kinase (MAPK) functional activity, rat lung lysates were immunoprecipitated with a monoclonal anti–phospho-ERK1/2 antibody. The captured proteins then were evaluated for kinase activity by measuring phosphorylation of Elk-1 protein, a substrate for ERK. As shown in Figure 4, significantly greater MAPK activity was detected in immunoprecipitated lung lysates from both groups of asbestos-exposed rats than in immunoprecipitates from sham-exposed rats.



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  		Figure 4. Western blots for MAPK functional activity in lung protein lysates from asbestos-exposed and sham-exposed rats. The lung lysate proteins were immunoprecipitated with anti–phospho-ERK1/2 antibody and then incubated with Elk-1 fusion protein in the presence of ATP. Samples then were loaded onto SDS-PAGE and subjected to Western blotting with anti–phospho-Elk1 antibody (A). Quantitation was performed by densitometric analysis (B). Positive control: purified ERK2 kinase. n = 3 rats per exposure group. *P < 0.05 versus sham-exposed group.

 
Phosphorylated ERK Proteins Coimmunoprecipitated with Nitrotyrosine
Figure 5 shows that both ERK1 and ERK2 proteins were captured in lung lysates immunoprecipitated with anti-nitrotyrosine and, when comparisons were made between the three exposed rat groups, no differences were detected in the amount of total ERK1/2 proteins. However, Western blots demonstrated significantly greater phosphorylation of ERK1/2 proteins in the immunoprecipitates after both types of asbestos exposure than after sham exposure (Figure 5). Once again, the two asbestos-exposed rat groups did not differ significantly from each other in this regard. The protein bands in Figure 1 provide confirmation that nitrated proteins actually were captured when the lung lysates were immunoprecipitated with anti-nitrotyrosine.



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  		Figure 5. Western blots for total ERK1/2 and phosphorylated ERK1/2 proteins in nitrated lung protein immunoprecipitates from asbestos-exposed and sham-exposed rats. Lung lysate proteins were immunoprecipitated with anti-nitrotyrosine antibody, separated by SDS-PAGE, and transferred to membranes. They then were blotted with anti–pan-ERK (A) or anti–phospho-ERK1/2 antibodies (B) and quantified by densitometric analysis (C). n = 3 rats per exposure group. *P < 0.05 versus sham-exposed group.

 
Both Long-Term and Short-Term Asbestos Inhalation–Induced Enhanced Immunoreactivity for Phospho-ERK and Nitrotyrosine within Lung Cells
The majority of the alveolar macrophages, as well as many of the bronchiolar epithelial cells and cells lining alveolar duct bifurcations, in lung sections from crocidolite-exposed rats showed intense cytoplasmic immunoreactivity for nitrotyrosine (Figure 6A). A similar pattern of strong immunostaining for nitrotyrosine was observed in lung sections from rats exposed either short-term (5-h) or long-term (2-wk) to chrysotile asbestos (Figure 6B). In contrast, only occasional alveolar macrophages and isolated bronchial epithelial cells demonstrated immunoreactivity for nitrotyrosine in lung sections from sham-exposed and carbonyl iron–exposed rats (Figure 6C). Immunospecificity of the anti-nitrotyrosine antibody was confirmed when immunostaining of the positive control ONOO--treated RPMC (Figure 6D) was abolished by absorption of the primary antibody with 3-nitro-L-tyrosine (Figure 6E) but not with O-phospho-L-tyrosine (result not shown). Moreover, immunoreactivity of the positive control cells was inhibited by reduction of nitrotyrosine to aminotyrosine with sodium dithionite (result not shown), confirming that nitrotyrosine truly was being detected by the primary antibody.







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  		Figure 6. Rat lung sections demonstrating immunohistochemical staining for nitrotyrosine. (A) Lung section from a crocidolite-exposed rat showing strong cytoplasmic immunostaining for nitrotyrosine in the airway epithelium and in the region of an alveolar duct bifurcation. Original magnification: x25. (B) Lung section from a rat exposed to chrysotile for 2 wk showing strong cytoplasmic immunostaining for nitrotyrosine in an alveolar macrophage, in the airway epithelium, and in the region of two alveolar duct bifurcations. Original magnification: x25. (C) Lung section from a carbonyl iron–exposed rat showing focal cytoplasmic immunostaining for nitrotyrosine in an occasional alveolar macrophage (red arrow). Another alveolar macrophage, which shows negative immunoreactivity for nitrotyrosine, contains numerous carbonyl iron particles (black arrow). Original magnification: x25. (D) ONOO--treated RPMC show strong cytoplasmic immunoreactivity for nitrotyrosine (positive control). Original magnification: x25. (E) Immunostaining of ONOO-–treated RPMC was abolished after absorption of the anti-nitrotyrosine antibody with 3-nitro-L-tyrosine, confirming the immunospecificity of the primary antibody. Original magnification: x25.

 
Focal groups of bronchiolar epithelial cells also displayed prominent immunoreactivity for phospho-ERK1/2 proteins in lung sections from crocidolite-exposed rats (Figure 7A) and from both groups of chrysotile-exposed rats (Figures 7B and 7C). Furthermore, strong immunoreactivity for phospho-ERK1/2 was identified in many alveolar macrophages from these animals. Although immunostaining for ERK proteins was mainly located within the cytoplasm of immunoreactive cells, intranuclear staining also was evident in some cells. In contrast with the effects of asbestos exposure, immunoreactivity for phospho-ERK1/2 was a rare occurrence in lung sections from either sham-exposed (Figure 7D) or carbonyl iron–exposed rats (Figure 7E). A key question as to whether or not phospho-ERK colocalized with nitrated proteins in the same lung cells could not be answered in this study because dual labeling within individual cells was difficult to interpret satisfactorily when lung sections were immunostained using two different chromogens (results not shown).







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  		Figure 7. Rat lung sections demonstrating immunohistochemical staining for phospho-ERK1/2. (A) Lung section from a crocidolite-exposed rat showing strong cytoplasmic immunostaining for nitrotyrosine in focal groups of airway epithelial cells. Original magnification: x25. (B) Lung section from a rat exposed to chrysotile for 5 h showing strong cytoplasmic immunoreactivity for phospho-ERK1/2 in the airway epithelium and in alveolar duct bifurcation cells. Original magnification: x25. (C) Lung section from a rat exposed to chrysotile for 2 wk showing strong cytoplasmic immunoreactivity for phospho-ERK1/2 in the airway epithelium and in four alveolar macrophages. Original magnification: x25. (D) Lung section from a sham-exposed rat showing absence of immunoreactivity for phospho-ERK1/2 in both alveolar macrophages and in the airway epithelium. Original magnification: x25. (E) Lung section from a carbonyl iron–exposed rat demonstrating cytoplasmic immunoreactivity for phospho-ERK1/2 in a solitary alveolar macrophage but not in the airway epithelium. Original magnification: x25.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There is evidence from experimental models of lung injury that mineral particles such as silica, coal dust, and asbestos can stimulate the formation of reactive nitrogen species in lung and pleural cells. Thus, when silica or coal dust was administered by intratracheal instillation to rats, enhanced iNOS gene expression and •NO formation were noted in BAL macrophages (24, 25) and in airway epithelial cells (24). The limited information that is available from clinical studies also suggests that patients with silicosis have upregulated •NO formation (25). Inhaled asbestos fibers generated similar effects to those of silica particles in rat alveolar and pleural macrophages (17, 19). Furthermore, we showed previously that inhalation of either chrysotile or crocidolite asbestos fibers induced the formation of nitrotyrosine within alveolar macrophages, airway epithelial cells, and visceral and parietal pleural mesothelial cells, with significantly greater quantities of nitrotyrosine being detected in lung extracts from asbestos-exposed rats than in sham-exposed rats (19).

Tyrosine nitration in tissues represents a footprint of the generation of reactive nitrogen oxides in vivo. Although ONOO- is considered to be a central contributor to the nitration of tyrosine residues (26), probably through the formation of intermediates generated by the reaction of ONOO- with CO2 (27), there is also evidence that alternative mechanisms of biologic nitration possibly may be operative in vivo. These include the catalytic oxidation of •NO and/or its metabolite NO2- by myeloperoxidase- and eosinophil peroxidase–dependent pathways, the reaction of •NO with tyrosyl radical, and the action of •NO2 radical (28–30). Irrespective of which nitration pathways may be involved, there is evidence that nitration of critical tyrosine residues may adversely affect the functional activity of certain proteins. For instance, in one study, addition of ONOO- to Mn superoxide dismutase (SOD) induced inactivation of enzymatic activity with targeted nitration of tyrosine residues (31). Notably, Tyr34, which is present in the active site of Mn SOD, was found to be especially susceptible to ONOO--mediated nitration. Other investigators have demonstrated that ONOO-–induced nitration of cytoskeletal proteins can disrupt the barrier function of endothelial cells (32) and inhibit the assembly of neurofilament subunits (33). Moreover, tyrosine nitration by either ONOO- or tetranitromethane was shown to impair the ability of surfactant protein-A to aggregate lipids, bind mannose, and to serve as a ligand for the adherence of Pneumocystis carinii to alveolar macrophages (34).

There is limited evidence that nitration of tyrosine residues may modify specific enzymatic function in vivo. In one study, analysis of nitrated proteins in kidneys from patients experiencing chronic allograft rejection revealed that Mn SOD was a target of tyrosine nitration, a finding that was associated with significantly decreased Mn SOD enzymatic activity (35). Another study showed that nitrated plasma proteins from patients with acute lung injury manifested significant impairment of ceruloplasmin ferroxidase activity and {alpha}1 protease inhibitor elastase-inhibiting activity (36). Furthermore, in a murine experimental model of Parkinson's disease, nitration of striatal tyrosine hydroxylase, the rate-limiting step for the synthesis of catecholamines, was associated with significant reduction of tyrosine hydroxylase activity and of striatal catecholamine levels (37).

The present study was undertaken to ascertain whether tyrosine nitration could be identified in specific lung proteins after asbestos inhalation and, if such were the case, whether that might affect the functional activity of the targeted proteins. We detected several protein bands ranging in size from {bsim} 35–50 kD in whole lung lysates from both asbestos-exposed and sham-exposed rats that were immunoprecipitated with anti-nitrotyrosine antibody. Of these, two distinct bands were readily detectable in lung lysates from crocidolite- and chrysotile-exposed rats, but not in sham-exposed animals. Notably, these protein bands were in the molecular size range of ERK1 and ERK2, respectively. Western blot analyses confirmed the presence of ERK1 and ERK2 proteins in whole lung as well as BAL cell lysates from all three groups of rats, and no differences were noted between exposure groups with respect to total ERK proteins in those lysates. However, both crocidolite and chrysotile inhalation induced significantly greater phosphorylation and functional activation of ERK proteins in lung cell lysates than was observed after sham exposure. Furthermore, BAL cell lysates from both groups of asbestos-exposed rats also demonstrated enhanced ERK signaling.

It is especially noteworthy that, when Western blots were performed with anti–phospho-ERK1/2 antibody on lung lysates immunoprecipitated with anti-nitrotyrosine antibody, phosphorylated ERK proteins coimmunoprecipitated with nitrotyrosine. Again, significantly greater ERK phosphorylation was detected in the nitrated immunoprecipitates from crocidolite- and chrysotile-exposed rat lungs than was observed after sham exposure. There was also enhanced immunoreactivity for phospho-ERK1/2 proteins and nitrotyrosine detectable in the majority of alveolar macrophages and airway epithelial cells in lung sections of asbestos-exposed rats, but these were rare occurrences in the lungs of sham-exposed and carbonyl iron–exposed animals, despite the fact that many carbonyl iron particles were readily detectable within rat lung cells. Notably, both long-term (2-wk) and short-term (5-h) chrysotile asbestos exposures were associated with increased immunostaining for nitrotyrosine and phospho-ERK1/2 within rat lung cells. Collectively, these immunohistochemical findings suggest that tyrosine nitration and enhanced ERK phosphorylation were effects that were unique for inhaled amphibole and serpentine asbestos fibers.

Our finding of enhanced ERK activation in the captured lung proteins immunoprecipitated with anti-nitrotyrosine antibody was unexpected, in view of previously mentioned studies that suggested that nitration of tyrosine residues was likely to produce decreased rather than enhanced enzymatic activity. Although this study did not address the mechanism of how asbestos-induced tyrosine nitration might have caused MAPK activation, we showed previously that asbestos fibers can induce ONOO- in rat pleural mesothelial cells (18), and there is evidence that ONOO- can trigger activation of ERK, p38 kinase, and c-Jun NH2-terminal kinase (JNK) cascades in vitro (38–41). It is known that ONOO- can activate the ERK cascade by targeting the epidermal growth factor receptor (EGFR), Raf-1, and MAPK kinase/ERK kinase (MEK-1), although precisely how ONOO- induces ERK activation has not been elucidated fully. Thus, one study demonstrated that ONOO- induced nitration and autophosphorylation of MEK-1 and that MEK-1 (but not EGFR or Raf-1) activation was required for ONOO--mediated ERK activation (40). In contrast, another study revealed that ONOO- induced ERK1/2 phosphorylation via a MEK-independent, calcium-dependent PKC pathway (41). Although either mechanism might explain how in vivo asbestos exposure could induce ERK activation, the fact that phospho-ERK1/2 proteins were coimmunoprecipitated with nitrotyrosine in the current study suggests the possibility that asbestos-induced ERK nitration actually may cause direct ERK autophosphorylation. Strong support for this notion is provided by a previous in vitro study that employed a cell-free system that excluded other proteins to show that ONOO- can directly induce MEK-1 autophosphorylation (40). In that study, ONOO- induced both MEK-1 nitration and MEK autophosphorylation in a concentration-dependent manner. It also is conceivable in the present study that asbestos exposure may have induced persistent ERK1/2 phosphorylation via inhibition of MAPK phosphatase-1 (MKP-1), because that phosphatase has been shown to dephosphorylate and inactivate ERK1/2 (42).

In summary, this study has demonstrated, in a rat in vivo model, that both serpentine and amphibole asbestos fibers induced protracted phosphorylation of intrapulmonary ERK proteins and upregulated Elk-1 phosphorylation by ERK. Furthermore, ERK phosphorylation was associated with tyrosine nitration, and the majority of alveolar macrophages and airway epithelial cells from asbestos-exposed rats manifested strong immunoreactivity for both phospho-ERK1/2 and nitrotyrosine. Because in different circumstances the ERK pathway is critical for both proliferative and cytotoxic responses (40), prolonged stimulation of the ERK signaling cascade may have importance to diverse asbestos-related cell-cycle alterations, including proliferation and repair in lung fibrogenesis as well as airway epithelial apoptosis and proliferative lesions.


   Acknowledgments
 
This study was supported by grant # HL-54196 from the National Institutes of Health and grant # MDA905-01-1-0001 from the US Department of Defense. The authors thank Dr. Jing-Lao Liu and Dr. Arnold Brody for generously providing archival rat lung tissue for immunohistochemical studies.

Received in original form January 31, 2002

Received in final form July 26, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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