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Am J Physiol Lung Cell Mol Physiol 292: L422-L429, 2007. First published October 6, 2006; doi:10.1152/ajplung.00204.2006
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Diesel exhaust particulate-induced activation of Stat3 requires activities of EGFR and Src in airway epithelial cells

Dongsun Cao,1 Tamara L. Tal,2 Lee M. Graves,3 Ian Gilmour,4 William Linak,4 William Reed,1 Philip A. Bromberg,1 and James M. Samet4

1Center for Environmental Medicine, Asthma and Lung Biology, 2Curriculum in Toxicology, and 3Department of Pharmacology, University of North Carolina, Chapel Hill; and 4Human Studies Division, United States Environmental Protection Agency, Research Triangle Park, North Carolina

Submitted 6 June 2006 ; accepted in final form 4 October 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
In vivo exposure to diesel exhaust particles (DEP) elicits acute inflammatory responses in the lung characterized by inflammatory cell influx and elevated expression of mediators such as cytokines and chemokines. Signal transducers and activators of transcription (STAT) proteins are a family of cytoplasmic transcription factors that are key transducers of signaling in response to cytokine and growth factor stimulation. One member of the STAT family, Stat3, has been implicated as a regulator of inflammation but has not been studied in regard to DEP exposure. The results of this study show that DEP induces Stat3 phosphorylation as early as 1 h following stimulation and that phosphorylated Stat3 translocates into the nucleus. Inhibition of epidermal growth factor receptor (EGFR) and Src activities by the inhibitors PD-153035 and PP2, respectively, abolished the activation of Stat3 by DEP, suggesting that Stat3 activation by DEP requires EGFR and Src kinase activation. The present study suggests that oxidative stress induced by DEP may play a critical role in activating EGFR signaling, as evidenced by the fact that pretreatment with antioxidant prevented the activation of EGFR and Stat3. These findings demonstrate that DEP inhalation can activate proinflammatory Stat3 signaling in vitro.

signal transducer and activator of transcription 3; epidermal growth factor receptor; reactive oxygen species; human airway epithelial cells; diesel exhaust particles

DIESEL EXHAUST PARTICULATES (DEP) emitted during the combustion of diesel fuel are a major contributor to airborne particulate matter (PM) mass in urban areas (7, 37, 50). DEP consist of a carbonaceous particle core onto which thousands of organic compounds can be adsorbed. DEP also contain notable quantities of noncarbonaceous inorganic species that are the result of small amount of fuel ash, ash components, and metallic additives present in lubrication oil and from engine wear. The organic compounds that are known to be proinflammatory or mutagenic include the polycyclic aromatic hydrocarbons (PAH), nitroaromatic compounds, quinones, aldehydes, heterocyclic compounds, and metal salts (10, 42). DEP emissions, which typically range from between 0.05 and 0.2 µm in diameter, can persist in the air and are readily inhaled and deposited throughout the respiratory tract. Exposure to DEP has been associated with numerous adverse health outcomes, including pulmonary inflammation, increased susceptibility to respiratory infections, increased risk of cancer, and exacerbation of asthma and chronic obstructive pulmonary diseases (32). Although the mechanisms responsible for DEP-induced lung health effects remain to be fully elucidated, inflammatory responses are thought to play a pivotal role.

Airway epithelial cells are known targets of PM inhalation and respond to DEP by producing numerous mediators involved in the lung immune and inflammatory responses, including production of cytokines, chemokines, and adhesion molecules (1, 4, 9, 51). The expression of such proinflammatory molecules is controlled at the transcriptional level through the activation of a network of intracellular signaling pathways that activate transcription activators, including NF-{kappa}B and AP-1 (19, 33, 48). DEP-induced activation of transcription factors is believed to be associated with the generation of reactive oxygen species (ROS) such as hydrogen peroxide, superoxide anion, and/or hydroxyl radical (38, 40).

Transcription factor activation induced by DEP-associated ROS production has been traditionally linked to the mitogen-activated protein kinase (MAPK) pathways (12, 19). Treatment of human airway epithelial cells (HAEC) with DEP leads to the phosphorylation-dependent activation of MAPK family signaling intermediates p38 MAPK (12, 19), which in turn activate the transcription factors NF-{kappa}B and AP-1 (19, 33, 48). These molecules bind to the promoter region of the genes encoding a variety of cytokines and chemokines, thereby increasing their expression (19). For example, interleukin 8 (IL-8), which is a potent attractant of neutrophils in the lung, is regulated by a combination of transcription factors that includes NF-{kappa}B, AP-1, and NF-IL6 in HAEC (28, 31, 44) and is induced by DEP. Functional binding elements for these transcription factors have been found in the IL-8 promoter region (39). Regulation of proinflammatory genes is not limited to those transcription factors, however. Recently, it was found that expression of IL-8 can be induced in lung epithelial cells through an NF-{kappa}B-independent mechanism (39). Moreover, Stat3, a member of the signal transducers and activators of transcription (STAT) family, has been shown to be an important transcriptional factor in the regulated expression of IL-8 and may be another important candidate for this effect (57).

STATs are a family of cytoplasmic transcription factors identified as being key mediators of physiological signaling by cytokines and growth factors (5, 6, 24). Upon activation by specific ligands, STATs are recruited to the cytoplasmic domain of the receptor, where they are activated by phosphorylation of conserved tyrosines by receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor (PDGFR) or by non-receptor kinases such as Src or Jak (17, 27, 35, 53). Phosphorylated STATs form either homodimer or heterodimers and then translocate to the nucleus, where they bind STAT-specific binding sites of target genes and activate gene expression. The seven STATs identified in mammals are involved in diverse cellular processes, including cell differentiation, proliferation, development, and apoptosis (24).

Recent evidence shows that Stat3 is involved in inflammatory responses. Activation of Stat3 plays an essential role in inflammatory bowel disease (IBD) and mediates IL-6 signaling in gut epithelial cells (46, 55). In a rat model of lung injury induced by IgG immune complexes, which has been used to study the role of chemokines and cytokines in the process of acute inflammation (22, 54), Stat3 activation was demonstrated in alveolar macrophages (15). Like the MAPK pathways, the JAK-STAT and phosphatidylinositol 3-kinase cascades are activated in response to the redox changes in cells (3, 23, 43). However, the exact mechanism for Stat3 activation is incompletely understood. In addition to cytokines and growth factors, ROS recently have been shown to mediate Stat3 signaling (26). Respiratory syncytial virus (RSV)-induced lung inflammation activates Stat3 in an ROS-dependent manner through a mechanism that does not involve the activity of Jak (26).

Although adverse health effects exerted by DEP have been associated with inflammation in lung airway epithelial cells, the molecular mechanism for these effects are not fully understood. In this study, we have shown that DEP exposure can induce Stat3 phosphorylation and nuclear translocation through a process that includes the generation of ROS and requires EGFR and Src activities, which play an essential role in this process in HAEC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Reagents. Tissue culture media and reagents were obtained from Cambrex (Boston, MA). Rabbit polyclonal phospho-specific antibody against p-Stat1, p-Stat2, p-Stat3, p-Src, and p-EGFR were purchased from Cell Signaling Laboratories (Boston, MA). Mouse monoclonal antibody against Stat1, Stat2, and Stat3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Src kinase inhibitor PP2 and EGFR inhibitor PD-153035 were obtained from Calbiochem (San Diego, CA). An Src kinase assay kit was purchased from Upstate (Lake Placid, NY). Butylated hydroxyanisole (BHA) was purchased from Sigma-Aldrich (St. Louis, MO).

Preparation of DEP. Two DEP samples were examined. The first, designated NIST-DEP, was obtained from the National Institute of Sciences and Technology (Donaldson, Minneapolis, MN). The material was collected using a diesel forklift and hot bag filter system. The diesel particulate material was collected in a 55-gallon drum, subsequently homogenized in a V-blender for 1 h, and stored in polyethylene bags. NIST-DEP contains 1.5% (wt/wt) extractable organic matter, and its ratio of organic carbon to elemental carbon (OC/EC) is 0.1. The certified analysis results of these particles are available online at http://patapsco.nist.gov/srmcatalog/certificates/2975.pdf. The second sample, designated C-DEP, was generated in June 2005 at the U.S. Environmental Protection Agency main campus (Research Triangle Park, NC) with the use of a 30-kW (40 hp) four-cylinder Deutz BF4M1008 diesel engine connected to a 22.3-kW Saylor Bell air compressor to provide a load. The engine and compressor were operated at steady state to produce 0.8 m3/min of compressed air at 400 kPa. This translates to ~20% of the engine's full-load rating. Emissions from the engine were diluted with filtered air (3:1) to near ambient temperatures (~35°C) and directed to a small baghouse (Dusyex model T6-3.5-9 150 ACFM pyramidal baghouse using a polyester felt bag). Gram quantities of C-DEP were collected from the baghouse using reverse air pulsing. Once collected, the C-DEP samples were stored in sealed containers in a refrigerator (~4°C). C-DEP contains 18.9% (wt/wt) extractable organic matter, and the ratio OC/EC is 0.35.

Cell culture and treatment. Human bronchial epithelial cells were obtained from normal adult volunteers by transbronchoscopic brushing of bronchi, following the protocol approved by the Committee on the Protection of the Rights of Human Subjects at the University of North Carolina. The isolated primary cells were cultured as previously described (41). In brief, 5 x 105 cells were seeded and grown to confluence in bronchial epithelial cell basal medium (BEBM) supplemented with 0.5 ng/ml human epidermal growth factor, 0.5 µg/ml hydrocortisone, 5 µg/ml insulin, 10 µg/ml transferrin, 0.5 µg/ml epinephrine, 6.5 ng/ml triiodothyronine, 50 µg/ml gentamicin, 50 ng/ml amphotericin-B, 52 µg/ml bovine pituitary extract, and 0.1 ng/ml retinoic acid on tissue culture plates coated with human collagen (Sigma) and then passaged two or three times in BEGM on ordinary tissue culture plates. Before treatment with DEP, cells were starved in BEBM without supplements for 12–16 h.

In vitro Src kinase assay. An in vitro Src kinase activity assay was performed according to the provided protocol (Upstate). After treatment with PP2 or DEP, cells were washed three times with cold PBS, lysed with lysis buffer [10 mM Tris·HCl (pH 7.6), 50 mM Na4P2O7, 50 mM NaF, 1 mM NaV3O4, 1% Triton X-100, and 1x protease inhibitor mixture tablet that included a broad spectrum of potent protein tyrosine phosphatases inhibitors; Roche, Basel, Switzerland], and scraped off the plate. The lysate was then centrifuged at 4°C, 14,000 rpm for 10 min. Supernatant was collected for protein quantitation. Protein quantitation was performed using protein assay solution (Bio-Rad, Hercules, CA) and BSA of known concentration as standards. Anti c-Src (Santa Cruz Biotechnology) was used to immunoprecipitate Src proteins. Protein G-agarose beads (20 µl; Santa Cruz Biotechnology) were added to the lysate and incubated for 2 h at 4°C with gentle agitation. The beads were collected by centrifugation at 4°C, 5,000 rpm for 1 min. The beads were resuspended and washed with lysis buffer three times. Immunoprecipitates were washed twice with Src buffer [100 mM Tris·HCl (pH 7.2), 125 mM MgCl2, 5 mM MnCl2, 2 mM EGTA, 250 mM sodium orthovanadate, and 2 mM DTT]. Src buffer containing 1 µg of the Src substrates and 10 µCi of [{gamma}-32P]ATP (3,000 Ci/mmol; Amersham) was added, and the reactions were incubated for 10 min at 30°C. Reactions were stopped by adding 20 µl of 40% TCA and then spotted onto p81 paper, followed by five separate washes with 0.75% phosphoric acid and then one wash with acetone for 3 min. The SRC activity, which is measured by the incorporation of [{gamma}-32P]ATP in substrate, was read by a scintillation counter.

EGFR dephosphorylation assay. Active EGFR (86 kDa) was induced to autophosphorylate by incubation at room temperature for 5 min in Mg2+-ATP cocktail. HAEC at ~80% confluency were starved overnight before treatment with 100 µg/ml NIST or vehicle control for 4 h and then harvested in a phosphatase lysis buffer containing 100 mM HEPES, 0.2% Nonidet P-40 (NP-40), 20 µg/ml PMSF, and 10 µM compound 56 (c56; Calbiochem). Cell lysates were normalized for protein content, and 200 µg of protein were brought up in 35 µl of phosphatase buffer composed of 25 mM HEPES (pH 7.2), 50 mM NaCl, and 2.5 mM EDTA. Harvested protein (100 µg) in 35 µl was added to the reaction mixture containing 115 µl of protein tyrosine phosphatase (PTP) buffer, 10 µM c56, and 20 µl of phosphorylated EGFR substrate (10 ng/µl), which was incubated at 30°C with mixing and sampled at 0, 5, 10, and 20 min. Each sample was placed in 15 µl of 4x loading buffer on ice, heated for 1 min at 100°C, and then subjected to SDS-PAGE and Western blotting as previously described to assess the change in phosphorylation over time.

Western blot analysis. Western blot analysis was performed as previously described (41) with minor modification. Briefly, cells were lysed with lysis buffer containing 50 mM Tris·HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, one tablet of proteinase inhibitor (Roche), 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS. The lysates were sonicated for 10 s and centrifuged, and supernatants were colleted. The protein concentration was determined using protein assay solution (Bio-Rad). The lysates were separated by electrophoresis in SDS-PAGE and then transferred to a nitrocellulose membrane (Bio-Rad). Western blots were probed with antibodies described above. The protein bands were detected using enhanced chemiluminescence (Amersham).

For preparation of cytoplasmic and nuclear extracts, cells were washed in cold PBS buffer, resuspended in 400 µl of 10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, and proteinase inhibitor and incubated on ice for 20 min. NP-40 was added to the cells to a final concentration of 0.5%, followed by centrifugation for 30 s at 4°C. Supernatant (corresponding to cytoplasmic extract) was collected. To obtain nuclear extracts, we resuspended the pellets in 50 µl of buffer containing 50 mM HEPES (pH 7.8), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 10% glycerol, and proteinase inhibitor (Roche). After vortexing for 20 min at 4°C, the suspension was centrifuged for 10 min and supernatant (nuclear extracts) was collected.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
DEP specifically induces phosphorylation of STAT3 in a time- and dose-dependent manner. Two types of DEP were used in this study, NIST and C-DEP. A major difference between these two samples is a log-fold difference in the fraction of extractable organic mass that they contain. NIST-DEP has ~2% extractable organic matter, whereas C-DEP contains ~20% extractable organic content. To investigate whether either DEP could induce the phosphorylation of STAT family transcription factors, we grew HAEC to confluence and deprived them of growth factors overnight before stimulation with 50 µg/ml DEP. No significant decrease in cell viability was found following treatment with up to 100 µg/ml of either DEP for 4 h (data not shown). With the use of phospho-site-specific antibodies recognizing p-Stat3, Western blotting showed that exposure of HAEC to either C-DEP or NIST-DEP can lead to phosphorylation of Stat3 in a dose- and time-dependent manner (Fig. 1). Elevated Stat3 phosphorylation was detectable at 1 h after treatment and increased up to 4 h following exposure. Stat3 was found to be dose-dependently phosphorylated in response to NIST-DEP treatment of HAEC at concentrations of 12.5–50 µg/ml, with higher doses inducing no additional strengthening of the p-Stat3 band (Fig. 1B). Neither DEP exposure induced phosphorylation of Stat1 (Fig. 1A) or Stat2 (data not shown). We also investigated the possibility of a delayed secondary effect on Stat1 activation by examining levels of p-Stat1 over a time course up to 24 h, using IFN-{gamma} as a positive control. DEP treatment did not result in a detectable activation of Stat1 in these cells (data not shown).


Figure 1
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  		Fig. 1. Diesel exhaust particulates (DEP) induce phosphorylation of Stat3 in vitro and in vivo. A: human airway epithelial cells (HAEC) were grown to confluence and stimulated with NIST-DEP (50 µg/ml) or C-DEP (50 µg/ml) for 1 and 4 h. Lysates were collected in RIPA buffer, and 50 µg of proteins were loaded for electrophoresis. The phosphorylation of Stat1 and Stat3 was detected using anti-phospho-Stat1 (pStat1) and anti-phospho-Stat3 (pStat3) antibodies. Anti-Stat1 and anti-Stat3 antibodies recognizing total Stat1 and Stat3 proteins were used to demonstrate equal loading. B: human primary cells were treated with varying concentrations of NIST-DEP as indicated for 4 h, and phosphorylation of Stat3 and total Stat3 were detected as in A.

 
Phosphorylated Stat3 is translocated into the nucleus in response to DEP exposure. Phosphorylation of Stat3 occurs in the cytoplasm and is followed by its dimerization and movement into the nucleus (5, 6, 21). To determine whether DEP-induced phosphorylation of Stat3 leads to its translocation in HAEC, we measured the levels of total and phosphorylated Stat3 in the cytoplasmic and nuclear compartments after exposure to 50 µg/ml NIST-DEP for 1–4 h by using Western blotting. As shown in Fig. 2, stimulation with NIST-DEP induced a time-dependent appearance of p-Stat3 in both cytoplasmic and nuclear compartments. The amount of total Stat3 detected in the cytoplasmic compartment decreased at 4 h, consistent with a translocation of the Stat3 protein into the nucleus (Fig. 2B). These data, taken together, suggest that Stat3, once activated by DEP, is translocated into the nucleus.


Figure 2
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  		Fig. 2. Phosphorylated Stat3 is translocated to nucleus. HAEC were grown to confluence and treated with NIST-DEP (50 µg/ml) for 1 and 4 h. Cytoplasmic and nuclear extracts were collected in RIPA buffer. A: 50 µg of cytoplasmic extracts were loaded for electrophoresis. The phosphorylation levels of Stat3 were detected using anti-phospho-Stat3 antibody. The total Stat3 level was detected using anti-Stat3 antibody. B: 25 µg of protein from nuclear extracts were loaded for electrophoresis. The phosphorylation levels of Stat3 were detected as in A.

 
DEP-induced activation of Stat3 is redox sensitive. DEP previously have been shown to induce ROS generation in lung epithelial cells and macrophages (26, 32). In addition, Stat3 activation has been reported to be associated with production of ROS in alveolar epithelial cells, since pretreatment of cells with the antioxidant BHA blocks the ROS-induced activation of Stat3 (26). To investigate whether ROS mediate DEP-induced activation Stat3, we treated airway epithelial cells with NIST-DEP in the absence or presence of BHA (400 µM) and detected the phosphorylation of Stat3 with an antibody specific for p-Stat3. As shown in Fig. 3A, the activation of Stat3 by NIST-DEP was reduced to basal levels by coadministration of the antioxidant BHA. To corroborate these findings, we further investigated whether oxidative stress in the form of H2O2 exposure could mimic DEP-induced Stat3 phosphorylation. Exposure to H2O2 induced a clear increase in the level of phosphorylation of Stat3 in HAEC (Fig. 3B).


Figure 3
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  		Fig. 3. Oxidative stress is involved in the activation of Stat3 induced by DEP. A: HAEC were treated with NIST-DEP (50 µg/ml) for 1 and 4 h in the presence or absence of the antioxidant butylated hydroxyanisole (BHA; 400 µM), and lysates were collected in RIPA buffer. Proteins (50 µg) were subjected to Western blot analysis. The activation of Stat3 was detected using anti-phospho-Stat3 antibody. B: H2O2 induced activation of Stat3. Methodology was as described in A except that H2O2 (100 µM) was used as stimulus instead of NIST.

 
EGFR and Src are involved in phosphorylation of Stat3. A number of receptor and non-receptor tyrosine kinases have been shown to phosphorylate Stat3 (6). Among them, the best studied are the JAKs and EGFR. JAKs are a family of tyrosine kinases that include Jak1, Jak2, Jak3, and Tyk2. To determine whether Stat3 phosphorylation is linked to DEP-induced JAK activation, we measured levels of p-Jak2 in HAEC by using Western blotting. No significant increases in p-Jak2 phosphorylation were detected within a 4-h period of exposure to 50 µg/ml NIST-DEP (Fig. 4A). Furthermore, an inhibitor of Jak2 activation, AG490, failed to block DEP-induced Stat3 phosphorylation at 4 h in HAEC (Fig. 4B). In contrast, the same exposure to NIST-DEP induced EGFR phosphorylation at Tyr-1068 and Tyr-845 in HAEC (Fig. 4C). In addition, the activation of EGFR could be blocked by the EGFR kinase activity inhibitor PD-153035 (Fig. 4C), which also completely blocked the activation of Stat3 induced by DEP (Fig. 4D). These data indicate that activation of Stat3 by DEP occurs through EGFR- but not JAK-mediated signaling.


Figure 4
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  		Fig. 4. Activation of Stat3 is dependent on the epidermal growth factor receptor (EGFR), not Jak2. HAEC were treated with NIST-DEP (50 µg/ml) for 1 and 4 h in the presence or absence of Jak2 inhibitor AG490 or EGFR inhibitor PD-153035. The lysates were collected in RIPA buffer. Proteins (50 µg) were subjected to Western blot analysis. The activation of Jak2, Stat3, and EGFR were detected using anti-phospho-Jak2 (pJak2; A), anti-phospho-Stat3 (B and D), and anti-phospho-EGFR antibody (pEGFR; C and E), respectively. E: antioxidant BHA (400 µM) blocked the activation of EGFR.

 
Because both ROS and EGFR play essential roles in activating Stat3 (Figs. 3 and 4, C and D), we further investigated whether DEP-induced EGFR can be abolished by antioxidant treatment. As shown in Fig. 4E, the antioxidant BHA (400 µM) prevented DEP-induced phosphorylation of the EGFR, suggesting that ROS is also a major stimulus for the phosphorylation of EGFR in HAEC exposed to DEP.

The non-receptor tyrosine kinase Src has been shown to be required for transducing EGFR signaling to Stat3 (35). To determine whether Src kinase activity is required in the phosphorylation of Stat3 induced by DEP exposure of HAEC, a similar pharmacological approach was used. Pretreatment with the Src kinase activity inhibitor PP2 for 30 min effectively blocked DEP-induced Stat3 phosphorylation (Fig. 5A). These data are consistent with a previous report that Src activity is required to transduce EGFR signaling to Stat3 (35). Interestingly, in both BEAS-2B (data not shown) and primary airway epithelial cells (Fig. 5B), Src was constitutively activated, as assessed by Western blotting using phospho-site-specific antibodies for Tyr-416, which is considered to be an indicator of the state of activation of Src. Confirming this finding, Src activity in HAEC was also found to be basally elevated in resting cells and could not be further increased in response to DEP treatment (Fig. 5C). Together, these data suggest that Src is required but not sufficient to activate Stat3 in response to DEP treatment in HAEC.


Figure 5
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  		Fig. 5. Src activity is required of EGFR to activate Stat3. HAEC were treated with NIST-DEP (50 µg/ml) for 1 and 4 h in the presence or absence of the Src inhibitor PP2, and lysates were collected in RIPA buffer. Proteins (50 µg) were subjected to Western blot analysis. The activation of Stat3 and SRC were detected by anti-phospho-Stat3 (A) and anti-phospho-Src antibody (pSRC; B). C: Src activity assay. Src was immunoprecipitated using anti-Src antibody, and Src activity was assayed using a Src activity assay kit.

 
Inhibiting tyrosine phosphatase activity induces Stat3 activation. Reversible redox inactivation of tyrosine phosphatases has been shown to play a pivotal role in the activation of growth factor signaling in a variety of cell types (13, 38). To determine whether activation of Stat3 by DEP exposure involves inhibition of tyrosine phosphatase activity, we measured Stat3 phosphorylation in HAEC treated with vanadate, a known phosphatase inhibitor. As shown in Fig. 6, vanadate induces Stat3 phosphorylation in HAEC. This effect can be prevented by concurrent administration of the antioxidant BHA, suggesting that ROS-induced inhibition of tyrosine phosphatase is involved in the activation of Stat3.


Figure 6
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  		Fig. 6. Inhibition of tyrosine phosphatase induces Stat3 phosphorylation. HAEC were treated with vanadate (100 µm) for 30 and 60 min in the presence or absence of antioxidant BHA (400 µm), and lysates were collected in RIPA buffer. Proteins (50 µg) were subjected to Western blot analysis. The activation of Stat3 was detected by anti-phospho-Stat3.

 
We recently reported that exposure to tyrosine phosphatase inhibitors reduces the rate of dephosphorylation of the EGFR in HAEC (49). To investigate whether DEP exposure induces a similar effect, we performed an in vitro EGFR dephosphorylation assay measuring the time-dependent dephosphorylation of exogenous p-EGFR in HAEC exposed to vehicle or NIST-DEP. As shown in Fig. 7, the p-EGFR dephosphorylation activity in HAEC exposed to DEP was markedly reduced relative to the activity in lysates prepared from vehicle-exposed controls. This effect was observed in the rates of EGFR-directed dephosphorylation activity toward phosphates at two sites, Tyr-1068 and Tyr-845, by 30 min (Fig. 7), suggesting that DEP inhibits EGFR-directed PTP activity in HAEC.


Figure 7
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  		Fig. 7. Exogenous EGFR dephosphorylation was inhibited in lysates obtained from HAEC exposed to NIST-DEP in vitro. HAEC were treated with 100 µg/ml NIST-DEP or vehicle control for 4 h. Active, phosphorylated EGFR substrate (1 ng/µl) was mixed with 200 µg of cellular lysate, and the reaction was sampled at 0, 30, 60, and 90 min. Lysates were analyzed for EGFR dephosphorylation over time via Western blot analysis with phospho-specific anti-EGFR antibodies. The results shown are representative of 3 or more experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
Inflammation is a central feature in many occupational and environmental lung exposures (11, 34, 37). Although increased synthesis of proinflammatory proteins underlies inflammation, the regulatory mechanisms responsible for proinflammatory gene expression in response to toxic exposures, including DEP, are largely unknown. Considerable attention has been focused on the activation of transcription factors, both NF-{kappa}B and AP-1, by environmental exposure (19, 33, 47, 48). However, relatively little is known about the role of other transcription factors in inflammation induced by agents such as DEP.

Stat3 has been implicated in a variety of chronic human inflammatory diseases, including rheumatoid arthritis (52), Crohn's disease, and ulcerative colitis (24). Stat3 also has been found to be involved in pulmonary inflammation induced by IgG IC in a mouse model (15). The role of Stat3 in the inflammatory effects induced by ambient air pollutants such as DEP, however, has not been investigated. We presently report that DEP activates Stat3 signaling after either exposure to cultured human airway epithelial cells. These findings offer a heretofore unrecognized pathway by which DEP may induce inflammatory responses in human epithelial cells.

A recent conceptual advance relevant to the understanding of DEP-induced lung inflammation is the recognition that redox regulation of protein phosphatases is a pivotal event in cellular signaling through growth factor receptors (13). Given that air pollutants such as DEP induce oxidant stress, DEP might act through dysregulation of signaling that is secondary to oxidant-induced inhibition of PTPs. There is evidence that organic compounds associated with DEP can generate cellular ROS directly or induce the cell to generate ROS intracellularly (25, 40). Among the redox-sensitive mechanisms, the MAPK signaling pathway has been implicated in DEP-induced inflammation (12, 19). Recently, Stat3 also has been demonstrated to be sensitive to changes in the redox state in a number of cell types. For example, H2O2 activates Stat3 and Stat1 in fibroblasts (43), and activation with angiotensinogen II, oxidized low-density lipoprotein, or hepatitis C virus NS5A is abrogated by the antioxidant treatment (16, 29, 30). Recently, RSV was found to be able to activate Stat3 in lung alveolar epithelial cells and is dependent on the ROS (26). In this report, we extend these results to the studies of DEP-induced inflammation in lung airway epithelial cells and explore the involvement of DEP-induced-ROS in activation of Stat3.

Although it has been observed that ROS are associated with RSV-induced activation of Stat3 (26), the intracellular signaling events that lead to ROS-induced Stat3 activation are poorly understood. Liu et al. (26) demonstrated that JAKs and Src kinases are not responsible for ROS-induced activation of Stat3 following RSV infection, because inhibition of their activities failed to block Stat3 activation. Other studies have shown that EGFR, a receptor tyrosine kinase reported to be involved in Stat3 activation (36), can be activated by ROS (2, 14) through a mechanisms that involves inhibition of tyrosine phosphatase activity (2, 8). In addition, inhibition of tyrosine phosphatase was observed following RSV infection, which is known to activate Stat3 (26). The involvement of EGFR in Stat3 signaling, however, was not examined in that study.

In the present study, we explored the possibility that EGFR is involved in Stat3 activation by DEP. Blocking EGFR kinase activity with PD-153035 blocked Stat3, directly implicating EGFR in DEP-induced induction of Stat3. We also observed that DEP-induced activation of EGFR could be blocked by antioxidant treatment, supporting the notion that ROS are involved in activation of EGFR. Given the involvement of ROS-induced PTP inhibition in the EGFR activation (2, 8), we speculate that oxidative stress induced by DEP may be responsible for the transactivation of EGFR seen in this study. Several lines of evidence have shown that Src is required for EGFR signaling in response to certain stimuli (35). Therefore, there is precedence that Src kinase activity is necessary to transmit the DEP-initiated signal through EGFR to Stat3. Remarkably, we did not find consistent activation of Jak2 in response to DEP exposure, and inhibition of Jak2 activity with AG490 did not blunt DEP-induced Stat3 activation (data not shown). However, this is consistent with a previous report showing that EGFR-mediated Stat3 activation is independent of Jak (26). Perhaps the most unexpected result of our study is that the basal levels of Src activity are constitutively active and cannot be further enhanced by DEP stimulation in cultured primary HAEC. Possibly, this basal activation of Src is an artifact of the conditions used to culture these cells. Nonetheless, given the fact that inhibiting Src activity abrogates the activation of Stat3, we conclude that Src activity is required but not sufficient to activate Stat3 in response to DEP.

Stat3 can play both positive and negative roles in the inflammatory and immune responses (18, 46, 55). DEP-induced activation of Stat3 may contribute positively to inflammatory reactions that are analogous to what has been discovered in IBD. In the case of IBD, upregulation of IL-6, a proinflammatory cytokine, and Stat3 were reported, and moreover, Stat3 was found to be responsible for mediating signaling elicited by IL-6 (20, 58). However, we were unable to detect either upregulation of IL-6 mRNA or the release of IL-6 protein in the medium within the 4-h treatment period of DEP (unpublished observations). Thus we believe that it is unlikely that observed Stat3 activation resulted from the IL-6-elicited signaling pathway. However, we cannot exclude the possibility of involvement of other cytokines. For example, IL-1, a cytokine that can activate Stat3 signaling, was found to be upregulated twofold by DEP within 4 h of treatment (unpublished observations). Stat3 also could play an inhibitory role in the immune response and function as a molecular in the cell-mounted cytoprotection against inflammation induced by DEP. Takeda et al. (45, 46) reported that mice with specific disruption of Stat3 in macrophage and neutrophil are susceptible to endotoxin shock with increased inflammatory cytokines such as TNF-{alpha}, IL-1, IFN-{gamma}, and IL-6. Hackenmiller et al. (18) also reported that knockout of a non-receptor tyrosine kinase (c-fes–/–), which is implicated in the cytokine receptor signal transduction, neutrophil survival, and myeloid differentiation, leads to increased expression of Stat3 and impaired immune response. Stat3 also has been shown to play an anti-inflammatory role as a mediator of the expression of the cytokine IL-10 (56). We have observed that DEP-treated HAEC exhibit a pronounced increase in production of IL-10 (unpublished data). Whether there is direct association between activation of Stat3 and IL-10 expression and its biological significance awaits further examination. Additional studies are required to investigate the role of Stat3 in the inflammatory effects of DEP inhalation.

In summary, we have demonstrated that DEP exposure can specifically activate the transcription factor Stat3 in primary cultures of human airway epithelial cells. We delineated the molecular signaling pathway for DEP-induced activation of Stat3 and found that ROS is a critical messenger that induces activation of EGFR via a mechanism that requires the activity of Src, which then induces the Stat3. ROS-induced activation of EGFR appears to be the result of DEP-induced impairment of EGFR-directed tyrosine phosphatase activity. These findings expand our understanding of the biochemical and molecular effects of DEP exposure of a relevant lung cell type and suggest an initiating mechanism for the toxicology of DEP inhalation.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
Portions of this work were performed under Contract EP-C-04-023 with ARCADIS G&M, Inc. The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory and National Risk Management Research Laboratory, U.S. EPA, and approved for publication. The contents of this article should not be construed to represent Agency policy nor does mention of trade names or commercial products constitute endorsement or recommendation for use.


   ACKNOWLEDGMENTS
 
We are grateful to Charly King, Daniel Janek, Todd Krantz, Mary Daniels, and Liz Boykin for considerable efforts in conducting the C-DEP exposure studies and to Dr. Jacqueline Bromberg, Robert Silbajoris, Lisa Daily, and Missy Brighton (Center for Environmental Medicine, Asthma and Lung Biology, University of North Carolina) for excellent technical assistance in the preparation and sectioning of embedded tissue during the health effects studies.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. M. Samet, Human Studies Division, 104 Mason Farm Rd., EPA Human Studies Facility, Chapel Hill, NC 27599-7315 (e-mail: samet.james{at}epa.gov)

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


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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