Ultrafine carbon black particles stimulate proliferation of human airway epithelium via EGF receptor-mediated signaling pathway
Jun Tamaoki,
Kazuo Isono,
Kiyoshi Takeyama,
Etsuko Tagaya,
Junko Nakata, and
Atsushi Nagai
First Department of Medicine, Tokyo Women's Medical University School of Medicine, Tokyo 162-8666, Japan
Submitted 28 June 2004
; accepted in final form 30 July 2004
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ABSTRACT
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Exposure to ambient ultrafine particles induces airway inflammatory reactions and tissue remodeling. In this experiment, to determine whether ultrafine carbon black (ufCB) affects proliferation of airway epithelium and, if so, what the mechanism of action is, we studied human primary bronchial epithelial cell cultures. Incubation of cells in the serum-free medium with ufCB increased incorporations of [3H]thymidine and [3H]leucine into cells in a time- and dose-dependent manner. This effect was attenuated by Cu- and Zn-containing superoxide dismutase (Cu/Zn SOD) and apocynin, an inhibitor of NADPH oxidase, and completely inhibited by pretreatment with the epidermal growth factor receptor (EGF-R) tyrosine kinase inhibitors AG-1478 and BIBX-1382, and the mitogen-activated protein kinase kinase inhibitor PD-98059. Transfection of a dominant-negative mutant of H-Ras likewise abolished the effect ufCB. Stimulation with ufCB also induced processing of membrane-anchored proheparin-binding (HB)-EGF, release of soluble HB-EGF into the medium, association of phosphorylated EGF-R and Shc with glutathione-S-transferase-Grb2 fusion protein, and phosphorylation of extracellular signal-regulated kinase (ERK). Pretreatment with AG-1478, [Glu52]Diphtheria toxin, a specific inhibitor of HB-EGF, neutralizing HB-EGF antibody, Cu/Zn SOD, and apocynin each inhibited ufCB-induced ERK activation. These results suggest that ufCB causes oxidative stress-mediated proliferation of airway epithelium, involving processing of HB-EGF and the concomitant activation of EGF-R and ERK cascade.
air pollution; epidermal growth factor receptor; mitogen-activated protein kinase; cellular proliferation
HIGH LEVELS OF AMBIENT RESPIRABLE pollutants have been linked to the increased risk for chronic lung diseases, including asthma and chronic obstructive pulmonary disease (1, 18, 27). Ultrafine particles are particles <100 nm in diameter and are derived from primary combustion sources, representing a variable and heterogeneous component of environmental particulate air pollution (10). A number of studies have suggested that ultrafine particles are particularly pathogenic compared with larger particles of the same mineral type and mass (12, 21, 25).
Ultrafine carbon black (ufCB), a very fine powdered form of elemental carbon, is manufactured by the controlled vapor-phase pyrolysis of liquid or gaseous hydrocarbons, and there is increasing evidence that ufCB particles induce intense inflammatory and immunomodulatory reactions in the airways through activation of neutrophils and alveolar macrophages (9,21, 30). However, it is also possible that inhaled ufCB could act directly on airway mucosal surface and produce functional and structural alterations of airway epithelial cells. Indeed, Churg et al. (6) have recently shown that a marked airway wall thickening can be seen in subjects exposed to high levels of particulate pollution. Therefore, the first objective of the present study was to determine whether ufCB has a growth-promoting activity in human airway epithelial cells in vitro.
Many growth stimuli cause tyrosine phosphorylation of various cellular proteins through receptors, either directly or indirectly coupled to tyrosine kinases, which are believed to play an essential role in mitogenesis and cell differentiation (36). Epidermal growth factor receptor (EGF-R) is a single-transmembrane receptor tyrosine kinase and, when tyrosine phosphorylated, provides binding sites for cellular proteins containing Src homology 2 domain of adaptor proteins, such as Shc and Grb2 (20, 36). Both EGF-R and Shc then bind to each other, as well as Grb2-Sos complex, thus enabling catalyzation of the conversion of P21ras-GDP to P21ras-GTP. The P21ras-GTP binds to and activates Raf-1 kinase and subsequently extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase kinase (MEK), which results in cell proliferation (23). However, the mechanism of growth-promoting effect of ufCB is unknown. Thus the second objective of the study was to elucidate a signaling pathway related to EGF-R in the proliferation of airway epithelial cells induced by ufCB.
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MATERIALS AND METHODS
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Reagents.
The following drugs and chemicals were used in the present experiments: apocynin, [Glu52]Diphtheria toxin, 1,10-phenanthroline (Sigma-Aldrich, St. Louis, MO), Cu- and Zn-containing superoxide dismutase (Cu/Zn SOD; Funakoshi, Tokyo, Japan), BIBX-1382 (Boehringer Ingelheim, Ingelheim, Germany), polyclonal anti-ERK antibody, polyclonal anti-phosphorylated ERK antibody, PD-98059 (New England Biolabs, Beverly, MA), monoclonal antiphospho-specific EGF-R antibody, polyclonal antiheparin-binding (HB)-EGF antibody (R&D Systems, Minneapolis, MN), agarose-conjugated glutathione-S-transferase (GST)-Grb2 (1127) fusion protein (Santa Cruz Biotechnology, Santa Cruz, CA), monoclonal antiphosphotyrosine antibody (RC20H; Transduction Laboratories, Lexington, KY), polyclonal anti-Shc antibody (Upstate Biotechnology, Lake Placid, NY), polyclonal antipro-HB-EGF antibody (Peptide Institute, Osaka, Japan), FITC-conjugated goat anti-rabbit IgG (Organon Teknika, Durham, NC), [3H]thymidine, [3H]leucine (NEN Life Science Products, Boston, MA), tyrphostin AG-1478 [4-(3-chloroanilino)-6,7-dimethoxyquinazoline], and tyrphostin AG-1296 (6,7-demethoxy-3-phenylquinoxaline; Calbiochem, La Jolla, CA). ufCB particles had a mean diameter of 11.2 ± 0.5 nm and surface area of 456.7 m2/g. Fine carbon black (CB) particles had a mean diameter of 250.4 ± 16.1 nm and surface area of 7.8 m2/g. These ufCB and CB were kindly provided by Tokai Carbon (Tokyo, Japan).
Preparation of human bronchial epithelial cells.
Normal human bronchial epithelial cells were prepared by the method reported previously (26). Briefly, normal human lobar or segmental bronchus was obtained either at the time of the resection of lung tumor or at autopsy (28 h postmortem). The bronchus was rinsed in sterile HBSS and incubated in Ham's F-12 medium containing 0.1% protease (type XIV, Sigma) at 4°C overnight. The bronchus was rinsed again, and the recovered cells were washed twice in HBSS. The study was planned according to the ethical guidelines following the declaration of Helsinki and given institutional approval.
Cell culture.
The cells were cultured in Lechner and La Veck media (LHC, which includes 0.72 ng/ml hydrocortisone and 1.24 ng/ml phenol red; Biofluids, Rockville, MD) on plates precoated with coating media containing 29 ng/ml collagen (vitrogen; Collagen, Palo Alto, CA), 10 µg/ml BSA (Biofluids) and 10 µg/ml fibronectin (Calbiochem) for 5 min. The cells were passaged at 6080% confluence by dissociation from plates with 0.02% trypsin (E-PET, Biofluids), which was neutralized with soybean trypsin inhibitor (SBTI, Biofluids). Primary bronchial epithelial cell cultures of passages 02 were used in the present study. In our preliminary experiment with primary bronchial epithelial cells and second-passaged cells, no less than 80% of the cells were positive for immunocytochemical staining with keratin but not with vimentin, indicating that the cells were of epithelial nature.
Proliferation of cultured epithelial cells.
DNA and protein syntheses in vitro were assessed by incorporation of [3H]thymidine and [3H]leucine into cells, respectively (16). The bronchial epithelial cells were plated in 100 µl of Ham's F-12 medium containing 10% FBS at a density of 104 cells per flat-bottomed well in 96-well microtiter plates and grown to subconfluence. After washing the cells three times with PBS, we arrested the cell growth by incubation for 72 h in Ham's F-12 medium without FBS. The medium was then replaced with serum-free medium, and the cells were incubated with ufCB or CB for up to 72 h in the absence or presence of the pharmacological blocking agents where indicated. After completion, the cells were incubated at 37°C for 4 h with [3H]thymidine or [3H]leucine at a final concentration of 1 µCi/ml, and trichloroacetic acid-insoluble radioactivity was measured in a liquid scintillation counter.
Transfection of a dominant-negative H-Ras mutant.
A replication-defective E1 and E3 adenoviral vector, containing CA promoter comprising a cytomegalovirus enhancer and chicken
-actin promoter, was ligated to a dominant-negative mutant of H-Ras (AdRasY57), in which tyrosine replaces aspartic acid at residue 57, as previously described (34). The epithelial cells grown in serum-free medium were incubated with AdRasY57 for 2 h, washed with fresh medium, and further incubated for 72 h in serum-free medium containing ufCB.
Immunocytochemistry.
The cells were plated onto eight-well tissue culture chamber slide (Lab Tek, Tokyo, Japan) at a density of 104 cells/100 µl medium containing 10% FBS. After 24 h, cells were replenished with serum-free medium and incubated for 72 h. Then, ufCB was added to the medium, and 5 min later the cells were fixed in 3% paraformaldehyde, permeabilized with 0.5% Triton-X, incubated in PBS containing 3% BSA and 1% goat serum, and stained with primary antibodies (1:50) against ERK (inactive form) and phosphorylated ERK (active form of ERK). Primary antibody was detected by FITC-conjugated secondary antibody (1:50), and the stained cells were observed under epifluorescence microscopy.
Immunoprecipitation.
For immunoblot analysis of EGF-R- or Grb2-associable proteins, cells were incubated with ufCB for the indicated times and lysed in 0.8 ml of lysis buffer (20 mM Tris·HCl, 150 mM NaCl, 2.5 mM EDTA, 1% Triton-X, 0.1% SDS, 10% glycerol, 50 mM NaF, 10 mM Na3P2O7, 1% deoxycholic acid, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin, pH 7.4). Lysates were sonicated and centrifuged at 14,000 g for 5 min, and the supernatant was rocked with polyclonal anti-EGF-R antibody (2 µg) with protein A/G agarose or agarose-conjugated GST-Grb2 fusion protein (3 µg) for 16 h at 4°C. Samples were centrifuged at 14,000 g for 5 s, and beads were washed three times with lysis buffer, solubilized in Laemmli sample buffer, and subjected to immunoblotting. After the membrane was initially treated with mouse monoclonal antiphosphotyrosine antibody (RC20H) (1:2,000), monoclonal antiphospho-specific EGF-R antibody (1:5,000), or polyclonal anti-Shc antibody (1:5,000), and then with secondary antibodies (1:2,000), immunoreactive proteins were detected by the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Tokyo, Japan).
For Western blot analysis, the cell lysates were solubilized in radioimmunoprecipitation assay buffer (0.15 M NaCl, 50 mM Tris·HCl, 0.5% Nonidet P-40, and 0.1% SDS) containing 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 1 mM sodium vanadate, then fractionated by SDS-PAGE, transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA), and probed with antibodies (1:1,000) against ERK and phosphorylated ERK. Primary antibody (1:2,500) was visualized using the ECL system (SuperSignal, Pierce, Rockford, IL).
ERK assay.
The epithelial cells, incubated for 72 h in serum-free medium, were stimulated with ufCB for 5 min at 37°C. The reaction was terminated by replacement of medium with ice-cold lysis buffer (10 mM Tris·HCl, 20 mM NaCl, 2 mM EGTA, 2 mM dithiothreitol, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) pH 7.4. After brief sonication (10 s), the samples were centrifuged for 5 min at 14,000 g, and the supernatant was assayed for ERK activity using an assay kit (Amersham Pharmacia Biotech) as described previously (16).
Release of HB-EGF.
HB-EGF is synthesized as a 20- to 30-kDa membrane-anchored HB-EGF precursor (pro-HB-EGF), and soluble HB-EGF is released as a paracrine growth factor. We therefore analyzed the processing of cell surface-associated pro-HB-EGF and the release of soluble HB-EGF by flow cytometry and Western blot analysis, respectively. For flow cytometry, cells were stimulated with ufCB for the indicated times and washed twice with 2 M NaCl in PBS to remove soluble HB-EGF trapped by the cell surface heparan sulfate proteoglycans. Then they were washed with ice-cold PBS and incubated for 30 min on ice with a rabbit antiserum against a synthetic peptide corresponding to amino acid 5473 of the human HB-EGF precursor. After the incubation, cells were washed twice with ice-cold PBS, and then FITC-conjugated goat anti-rabbit IgG was added. After a 30-min incubation on ice, fluorescence intensity of the cell surface was analyzed using a FACScan (Becton Dickinson, Mountain View, CA). Cytometric analyses were made on a logarithmic scale, and mean fluorescence intensity was converted to a linear scale to calculate the relative fluorescence intensity.
For analysis of the release of soluble HB-EGF, cells were stimulated with ufCB, and the supernatant of the conditioned medium was applied to heparin-Sepharose CL-6B column (Pharmacia, Uppsala, Sweden). After being washed with 10 mM Tris·HCl, heparin-Sepharose beads were suspended in SDS-PAGE sample buffer (25 mM Tris·HCl, 2% SDS, 10% glycerol, and 0.05% bromphenol blue). The supernatants were then fractionated by SDS-PAGE, the proteins were transferred to polyvinylidene difluoride membrane, and Western blot analysis was performed using anti-HB-EGF antibody.
Statistics.
All values were expressed as means ± SE. One-way analysis of variance and Newman-Keuls multiple-comparison test were used to determine statistically significant differences between groups, and P < 0.05 was considered significant.
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RESULTS
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ufCB-induced proliferation of airway epithelium.
Human bronchial epithelial cells that had been incubated for 72 h in Ham's F-12 medium without FBS did not proliferate during the next 72 h in the serum-free medium. However, addition of ufCB, at a concentration of 50 µg/ml, which corresponded to a particle density of 30.7 µg/cm2, to the serum-free medium stimulated DNA synthesis and protein synthesis, where incorporations of [3H]thymidine and [3H]leucine into cells compared with controls were 264 ± 33 and 222 ± 26%, respectively (P < 0.01, n = 6 for each), at 72 h after initiation of the culture. In contrast, the same dose of CB, a larger particle with a similar chemical composition, had little effect (Fig. 1). The potency of ufCB in cell proliferation was similar to that of 10% FBS. As shown in Fig. 2, ufCB caused dose-dependent increases in [3H]thymidine and [3H]leucine incorporations, the ED50 values being 14.4 ± 1.8 µg/cm2 for DNA synthesis and 15.3 ± 2.5 µg/cm2 for protein synthesis (n = 5 for each). This growth-promoting effect was greatly inhibited by the addition of Cu/Zn SOD (150 U/ml) and apocynin (30 µM), an inhibitor of NADPH oxidase (29). In a separate experiment, incorporations of [3H]thymidine and [3H]leucine after a 72-h incubation with 10% FBS were reduced by only 2 ± 3 and 4 ± 6%, respectively (n = 4), by Cu/Zn SOD, and 6 ± 5 and 8 ± 8%, respectively (n = 5), and by apocynin, indicating that the suppression of ufCB effect by these inhibitors is not due to toxicity or to a nonspecific effect.

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Fig. 1. Time course of the effect of ultrafine carbon black (ufCB, 30.7 µg/cm2) on DNA synthesis (top) and protein synthesis (bottom) in human bronchial epithelial cells cultured in serum-free medium. After completion of culture, incorporations of [3H]thymidine and [3H]leucine were measured. For control experiment, the cells were cultured in serum-free medium without any treatment. In some experiments, the cells were cultured in the medium containing 10% FBS or the medium containing carbon black (CB, 30.7 µg/cm2). Data are means ± SE; n = 6 for each point. *P < 0.05, **P < 0.01, significantly different from control values. dpm, Disintegrations/min.
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Fig. 2. Concentration-dependent effect of ufCB in the absence and presence of apocynin (30 µM, NADPH oxidase inhibitor) or Cu- and Zn-containing superoxide dismutase (Cu/Zn SOD, 150 U/ml) on DNA synthesis (top) and protein synthesis (bottom) in human bronchial epithelial cells cultured in serum-free medium. The cells were incubated with ufCB for 72 h, and incorporations of [3H]thymidine and [3H]leucine were measured. Data are means ± SE; n = 5 for each point.
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To assess the involvement of EGF-R phosphorylation in ufCB-induced cell proliferation, the cells were incubated for 2 h with AG-1478 or BIBX-1382 (200 nM) and cultured for 72 h in the serum-free medium containing ufCB (30.7 µg/cm2). Moreover, to determine whether ERK cascade is involved, we tested the effects of adenovirus-mediated transfection of a dominant-negative mutant of H-Ras (AdRasY57) and the MEK inhibitor PD-98059 (100 µM) (2) in a similar manner. As shown in Fig. 3, DNA synthesis and protein synthesis induced by ufCB were completely inhibited by AG-1478, BIBX-1382, AdRasY57 transfection, and PD-98059 each.

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Fig. 3. Effects of AG-1478, BIBX-1382, dominant-negative mutant of H-Ras (AdRasY57), and PD-98059 on ufCB-induced incorporations of [3H]thymidine (top) and [3H]leucine (bottom) into human bronchial epithelial cells. After a 2-h incubation with AG-1478 [200 nM, EGF receptor (EGF-R) tyrosine kinase inhibitor], BIBX-1382 (200 nM, EGF-R tyrosine kinase inhibitor), or PD-98059 (100 µM, MEK inhibitor), the cells were further incubated for 72 h with ufCB (30.7 µg/cm2). In some experiments, AdRasY57-transfected cells were likewise stimulated for 72 h with ufCB. For control experiment, the cells were cultured in serum-free medium without any treatment. Data are means ± SE; n = 6 for each column. **P < 0.01, significantly different from control values. P < 0.05,  P < 0.01, significantly different from corresponding values for ufCB alone.
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ufCB-induced ERK activation via transactivation of EGF-R.
Because the association of adaptor proteins (Shc and Grb2) with phosphorylated protein tyrosine kinases plays a role in recruiting Sos, an activator of p21ras, and thereby initiates sequential activation of ERK cascade (35), we examined whether ufCB stimulates the association of EGF-R and Shc with GST-Grb2 fusion protein. ufCB (30.7 µg/cm2) rapidly (within 2 min) increased association of tyrosine-phosphorylated 180-kDa protein with GST-Grb2 fusion protein, and the protein was identified as EGF-R, as recognized by anti-EGF-R antibody (Fig. 4). The rapid association of two Shc isoforms (p52 and p46) with GST-Grb2 fusion protein was also observed on stimulation with ufCB. These effects were inhibited by preincubation with [Glu52]Diphtheria toxin (10 µg/ml), a specific inhibitor of HB-EGF (24).

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Fig. 4. Effect of ufCB on association of tyrosine-phosphorylated EGF-R and Shc with Grb2 in human bronchial epithelial cells. After a 2-min stimulation of cells with ufCB (30.7 µg/cm2), cell lysates were coprecipitated (IP) with glutathione-S-transferase (GST)-Grb2 fusion protein and then immunoblotted with anti-EGF-R antibody (Ab, top) and anti-Shc antibody (bottom). In some experiments, the cells were incubated for 30 min with [Glu52]Diphtheria toxin (10 µg/ml) and then stimulated with ufCB.
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To determine whether stimulation with ufCB actually results in ERK activation, we studied immunocytochemistry and immunoblotting using an antibody that selectively recognizes enzymatically active Tyr204-phosphorylated ERK. Immunocytochemistry showed that ERK-like immunoreactivity was observed both in the ufCB (30.7 µg/cm2)-treated cells and the cells that had not been exposed to ufCB. In contrast, immunoreactivity for phosphorylated ERK was found only in ufCB-treated cells (Fig. 5). Western blot analysis shows that incubation of cells with ufCB caused phosphorylation of ERK that was detectable at 2 min, peaked at 5 min, and decreased at 10 min. As shown in Fig. 6, the effect of ufCB on ERK activation was as potent as 10% FBS and markedly inhibited by AG-1478 (200 nM), [Glu52]Diphtheria toxin (10 µg/ml), and neutralizing antibody for HB-EGF (100 µg/ml), but not by AG-1296 (10 µM), a specific inhibitor of PDGF-R kinase (19). The ufCB-induced ERK activation was also inhibited by apocynin (30 µM) and Cu/Zn SOD (150 U/ml).

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Fig. 5. Immunofluorescence staining of human bronchial epithelial cells for ERK (top) and phosphorylated ERK (p-ERK, bottom). After a 72-h incubation in serum-free medium, the cells were exposed to serum-free medium alone (control, A and C) or that containing ufCB (30.7 µg/cm2, B and D), and 5 min later immunostaining was performed. Bars indicate 5 µm.
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Fig. 6. Western blotting of ERK and p-ERK proteins (blots at top) and ERK activity (bottom) in human bronchial epithelial cells after a 5-min exposure to serum-free medium alone (control, lane 1), or that containing 10% FBS (lane 2) or ufCB (30.7 µg/cm2, lane 3). In some experiments, the cells were pretreated for 30 min with AG-1296 (10 µM, PDGF-R kinase inhibitor, lane 4), AG-1478 (200 nM, EGF-R tyrosine kinase inhibitor, lane 5), [Glu52]Diphtheria toxin [DT, 10 µg/ml, heparin-binding (HB)-EGF inhibitor, lane 6], neutralizing HB-EGF antibody (100 µg/ml, lane 7), apocynin (30 µM, NADPH oxidase inhibitor, lane 8), or Cu/Zn SOD (150 U/ml, lane 9), and then stimulated with ufCB. **P < 0.01, significantly different from control;  P < 0.01, significantly different from values for ufCB alone.
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ufCB-induced HB-EGF release.
As shown in Fig. 7, ufCB induced a rapid loss of cell surface-associated pro-HB-EGF, which began to recover in 20 min and then increased gradually in a time-dependent manner. In contrast, CB had no effect. In Western blot analysis, ufCB but not CB induced the appearance of immunoreactive mature HB-EGF in the conditioned medium. Two species of soluble HB-EGF (14 and 19 kDa) were detected within 20 min after the addition of ufCB. Pretreatment with apocynin (30 µM), Cu/Zn SOD (150 U/ml), and 1,10-phenanthroline (10 µM), a metalloproteases inhibitor (3), reduced the ufCB-induced release of HB-EGF.

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Fig. 7. Top: kinetics of cell surface pro-HB-EGF processing by ufCB. Human bronchial epithelial cells were incubated with 30.7 µg/cm2 ufCB or CB for the indicated periods, and cell surface pro-HB-EGF levels were measured. The absolute fluorescence intensities on flow cytometry were converted to relative values. Bottom: Western blot analysis of the released soluble HB-EGF into conditioned medium. The cells were incubated for 20 min in serum-free medium with 30.7 µg/cm2 ufCB or CB. The media were pooled and applied to a heparin-Sepharose CL-6B column. The proteins bound to heparin-Sepharose beads were fractionated by 15% SDS-PAGE and transferred to membranes. In some experiments, the cells were pretreated for 30 min with apocynin (Apo, 30 µM), Cu/Zn SOD (SOD, 150 U/ml), or 1,10-phenanthroline (Phenant, 10 µM, metalloproteases inhibitor), and then stimulated with ufCB.
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DISCUSSION
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Epidemiological studies have shown associations between exposure to ambient air particulate matter and adverse health outcomes, such as decreased pulmonary function and increased airway hyperresponsiveness and exacerbations of asthma (1, 18, 27). Among a wide variety of environmental particulate air pollutants, ultrafine particles, particularly ufCB, have received considerable attention in relation to their potential role in the induction of airway inflammation and the resultant tissue remodeling in the lungs (9, 21, 30). Thickening of airway wall is one of the characteristic findings of airway remodeling in asthma that may relate to hyperplasia of airway mucosal cells, fibroblasts, and smooth muscle cells. This change in the airway wall likely contributes to the development of bronchial hyperresponsiveness and irreversible bronchoconstriction, because the thickening gives rise to exaggerated decrease in airway caliber when the smooth muscle shortens (17). Airway epithelial cells are one of the first targets for ambient pollutants, and, in our study, incubation of primary culture of human bronchial epithelial cells with ufCB but not CB increased incorporations of both [3H]thymidine and [3H]leucine even in the culture medium from which FBS was removed to arrest the cell growth. This finding indicates that ufCB can stimulate DNA and protein syntheses, which may result in the thickening of airway wall.
The ineffectiveness of fine CB particles compared with ultrafine particles indicates that the observed effect is probably associated with small particle sizes and large surface area, as has been reported with other experimental systems (12, 21, 25). In addition, ultrafine particles are known to be potent inducers of intracellular oxidant generation (5, 22). In accordance with this notion, we found that pretreatment of cells with Cu/Zn SOD and the NADPH oxidase inhibitor apocynin (29) inhibited the growth-promoting response to ufCB, suggesting a role of oxidative stress in our experimental condition.
It has been generally accepted that EGF-R serves as a point of convergence for mitogenic signals arising from diverse stimuli. EGF-R transactivation can follow activation of cytokine receptors (37) and G protein-coupled receptors (14). For example, Daub and coworkers (7) have demonstrated that certain G protein-coupled receptor agonists, such as endothelin-1 and thrombin, induce tyrosine phosphorylation of EGF-R in rat fibroblast cell line and that treatment with an EGF-R tyrosine phosphorylation inhibitor or transfection of a dominant-negative EGF-R mutant suppresses ERK activation, c-fos expression, and DNA synthesis stimulated by these agonists. Furthermore, recent studies have shown that exposure of the human airway epithelial cell line NCI-H292 to cigarette smoke (32) and hydrogen peroxide (31) each upregulates mucin synthesis through EGF-R transactivation. Although recent evidence suggests that exposure to particulate air pollution may induce airway mucosal thickening (6) and proliferation of alveolar epithelial cells (33), involvement of EGF-R signaling in such a growth-promoting effect has not been studied. In the present experiment, ufCB-induced DNA synthesis and protein synthesis in human bronchial epithelial cells were almost completely inhibited by the EGF-R-specific tyrophostins AG-1478 and BIBX-1382. Moreover, the effects of ufCB were inhibited by adenovirus-mediated transfection of a dominant-negative mutant of H-Ras (AdRasY57) and the highly selective MEK inhibitor PD-98059 (2). Although PD-98059 has recently been shown to directly inhibit cyclooxygenase (4), the cyclooxygenase inhibitor indomethacin failed to alter the ufCB's growth-promoting effect (data not shown), suggesting that eicosanoid formation is not involved. Together, transactivation of EGF-R, with a concomitant activation of ERK cascade, may play a crucial role in the ufCB-induced proliferation of airway epithelium.
The Shc proteins have been implicated in p21ras-dependent ERK activation by several G protein-coupled receptor agonists (35). The present study clearly showed that ufCB rapidly increased the amounts of two Shc isoforms (p46 and p52) and tyrosine-phosphorylated EGF-R associated with GST-Grb2 fusion protein. Thus transactivation of EGF-R by ufCB causes recruitment and phosphorylation of Shc, thereby activating the ERK cascade. We also confirmed by immunocytochemistry, Western blot analysis, and ERK assay that incubation with ufCB actually induced ERK activation, and this effect was markedly inhibited by AG-1478 but not by the PDGF-R-specific tyrphostin AG-1296 (19). These data further suggest that EGF-R transactivation is a major signaling event for the ERK-related effect of ufCB. Moreover, the ufCB-induced phosphorylation of ERK was inhibited by Cu/Zn SOD and apocynin. This finding suggests that oxidative stress is upstream of ERK activation in the signal transduction pathway.
There is evidence that autocrine/paracrine release of soluble EGF-like ligands may account for EGF-R transactivation induced by certain stimuli (8, 11). For example, HB-EGF, a peptide mitogen of the EGF family, is released by proteolytic cleavage of a larger membrane-anchored precursor. In our experiment, Diphtheria toxin, which binds to the extracellular HB-EGF domain and specifically inhibits its mitogenic activity (24), potently attenuated the ufCB-induced association of Shc with GST-Grb2 fusion protein and activation of ERK, indicating an involvement of HB-EGF. We confirmed this by measuring the protein secretion of HB-EGF. HB-EGF is synthesized as a prepro-form, 208 amino acid long, expressed as a proform integrated into the plasma membrane, and then processed to a soluble form of 7687 amino acid residues through proteolytic cleavage (13). Our study showed that membrane-anchored pro-HB-EGF was decreased after treatment with ufCB and that this coincided with the occurrence of soluble forms of HB-EGF in conditioned medium. Therefore, shedding of HB-EGF may be operating. Previous studies have shown in certain experimental conditions that proteolysis of the HB-EGF precursor and cell surface shedding of HB-EGF are mediated by members of the ADAM family of metalloproteases (28) and that matrix metalloprotease can be released by the activation of NADH/NADPH oxidase (15). Indeed, we found that the ufCB-induced HB-EGF release was inhibited by Cu/Zn SOD, apocynin, and 1,10-phenanthroline. It is thus possible that oxidative stress-induced release of metalloproteases could have caused HB-EGF shedding.
In conclusion, we have demonstrated herein, for the first time, that ufCB stimulates proliferation of human airway epithelial cells. This growth-promoting effect is accounted for by autocrine release of HB-EGF and the subsequent EGF-R transactivation and ERK cascade activation.
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GRANTS
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This work was supported in part by Grant-in-Aid 06670243 from the Ministry of Education, Sports, Science, and Culture, Japan.
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ACKNOWLEDGMENTS
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The authors thank Masayuki Shino and Yoshimi Sugimura for their technical assistance. We also thank Peptide Institute, Inc. for providing us with antipro-HB-EGF antibody.
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FOOTNOTES
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Address for reprint requests and other correspondence: J. Tamaoki, First Dept. of Medicine, Tokyo Women's Medical Univ., 8-1 Kawada-Cho, Shinjuku, Tokyo 162-8666, Japan (E-mail: jtamaoki{at}chi.twmu.ac.jp)
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.
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