From the Biomedical Sciences Program, Cardiovascular Research Institute and Department of Anatomy, University of California, San Francisco, California 94143
Received for publication, July 12, 2002 , and in revised form, April 21, 2003.
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ABSTRACT |
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INTRODUCTION |
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However, conversion of adducts to mutations can only occur in proliferating cells (2, 3) suggesting that tobacco smoke must also promote cell division before carcinogens such as benzopyrenediol epoxide can effectively mutate DNA. Indeed, hyperproliferation occurs in response to smoke exposure (4, 5) and very likely increases cancer risk in the presence of tobacco carcinogens.
The mechanism by which tobacco smoke stimulates lung cell proliferation is unknown. Based on the involvement of the epidermal growth factor receptor (EGFR)1 in response to noxious stimuli (6, 7), we tested and confirmed the involvement of EGFR in the response of the host cell to tobacco smoke (8). In the experiments reported here, we identify mechanisms triggered by smoke that result in both phosphorylation (activation) of EGFR and cell proliferation.
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MATERIALS AND METHODS |
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MiceSix specific pathogen-free C57 black mice (8 weeks old, Charles River Laboratories, Wilmington, MA) were treated intranasally with the metalloproteinase inhibitor GM6001 or saline solution prior to exposure to smoke in a specially designed chamber (9).
Cell Culture and Assay for Cell DensityPrimary human airway epithelial cells were obtained from Clonetics (San Diego, CA) and were cultured under conditions recommended by the vendor. NCIH292 (mucoepidermoid carcinoma) cells were also used. NCIH292 cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin. For cell proliferation assays, cell density was assayed 24 or 48 h after a 4-h treatment with serum-free medium (SFM), smoke-containing medium, or EGF using Cell Titer 96® Aqueous One reagent (Promega, Madison, WI) according to the manufacturer's instructions.
Immunoprecipitation and ImmunoblottingCells were lysed in 20 mM Tris-HCl, 150 mM NaCl, 0.5% Triton X-100, 0.1% SDS, 1 mM EDTA, and 1 mM sodium orthovanadate. The samples were pre-cleared by centrifugation at 10,000 rpm for 10 min at 4 °C, and total protein concentrations were determined using the Bradford protein assay (Bio-Rad). For detection of ADAM (a disintegrin and metalloproteinase) proteins, we used lysates from cells that had or had not been transfected with morpholino antisense oligonucleotides. Lysis buffer contained 10 mM 1,10-ortho-phenanthroline to prevent autolysis of the ADAMs. For detection of EGFR ligands shed into cell culture medium, we concentrated the medium 10x using Amicon Centriplus filters with a cutoff of 3 kDa. For determination of the phosphorylation state of EGFR, we incubated equal amounts of lysate with anti-EGFR antibody and Protein A-agarose beads overnight at 4 °C. The lysate-antibody-bead complex was spun down and washed three times with lysis buffer. Following the final wash, 40 µl of SDS gel-loading buffer was added, the mixture was heated at 100 °C for 3 min, and proteins were resolved by SDS-PAGE. For immunoblot analysis of the samples listed above, proteins were transferred to nitrocellulose membranes using the Bio-Rad Mini Trans-Blot electrophoretic transfer cell. Membranes were blocked for 1 h at room temperature in phosphate-buffered saline containing 0.1% Tween 20 (PBS/Tween) and supplemented with 5% BSA, then washed with PBS/Tween and incubated with the appropriate antibody overnight at 4 °C. After removing primary antibody with several washes of PBS/Tween, the blot was placed in the appropriate horseradish peroxidase-conjugated secondary antibody for 45 min. After several washes, the antibody-antigen complexes were visualized using the ECL chemiluminescence detection system (Amersham Biosciences).
Smoke ExposuresSmoke particulates were generated in specially designed animal exposure chambers operated by Dr. Kent Pinkerton at the University of California, Davis (9). Pall Gelman Pallflex® borosilicate filters (Fisher) were inserted in line during the operation of the chambers. The total suspended smoke particles deposited on each filter were calculated by weighing filters before and after smoke exposure. Filters were mailed to our laboratory at the University of California, San Francisco, where they were stored at 4 °C until use. Prior to each experiment, we incubated filters in a volume of SFM to provide a final concentration of 1.0 mg of smoke particulate per ml of SFM. The filters were rotated in this medium at 4 °C for 3 days prior to use.
Detection of Reactive Oxygen Species (ROS) ProductionNCIH292 cells were incubated for 30 min prior to smoke or SFM exposure with 10 µM 2',7'-dichlorodihydrofluorescein diacetate, H2DCFDA (Molecular Probes, Eugene, OR). Fluorescent images were captured using a Nikon Eclipse E600 microscope equipped with epifluorescence optics and a Zeiss Axiocam digital camera.
ImmunofluorescenceNCIH292 cells grown on coverslips were incubated for 15 min at 37 °C with or without smoke-containing medium or EGF (100 ng/ml). Cells were then fixed, permeabilized, and immunostained with anti-EGFR antibody (Santa Cruz Biotechnology, Inc.) and fluorescein isothiocyanate-conjugated sheep anti-mouse IgG (Amersham Biosciences) prior to viewing with a Nikon Eclipse E600 microscope using an NCF Fluor 40 objective lens.
Morpholino Antisense OligonucleotidesNCIH292 cells were
transfected according to the manufacturer's instructions with 2
µM solutions of morpholino antisense oligonucleotides (Gene
Tools LLC, Philomath, OR) corresponding to ADAM10,
5'-AATTAACACTCTCAGCAACACCATC-3'; ADAM15,
5'-AGAGCAGCGCCAGCCGCATGGCAGC-3'; ADAM 9,
5'-GAAAGCGCGCGCCAGACCCCATCTC-3'; or ADAM 17,
5'-TCAGGAATAGGAGAGACTGCCT-3'). Thirty h later, cells were lysed
for ADAM immunoblot or stimulated with smoke or EGF. ADAM immunoblots were
normalized for loading differences using -actin and analyzed by
densitometry (Image J 1.27 software). Stimulated cells were used for
immunoprecipitation and phospho-EGFR immunoblot as described above.
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RESULTS |
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Although there have been prior reports of "ligand-independent" EGFR activation, the mechanism of activation is unclear. Following smoke exposure, we observed oligomerization and internalization of EGFR (Fig. 2C) with the same kinetics as that seen following administration of EGF itself (not shown). Oligomerization implies that smoke induces autophosphorylation of the receptor, a mode of activation inhibitable by AG 1478. It has only recently been appreciated that receptor activation in the absence of exogenous growth factor can nonetheless be growth factor-dependent. Thus, Ullrich and co-workers (10) showed that the transactivation of EGFR by G protein-coupled receptors was mediated by the metalloproteinase-dependent cleavage of HBEGF and its subsequent binding to EGFR. We recently showed that transactivation of EGFR by the bacterial outer membrane component LTA occurs by a similar mechanism (11). To examine the mechanism induced by smoke, we first asked whether ligand binding was required for the response. Results showing attenuation of receptor phosphorylation by antibody blockade of the ligand-binding site indicated that despite the absence of an extracellular ligand, ligand-receptor interaction was required for the response (Fig. 3A).
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Because EGFR ligands (amphiregulin, betacellulin, EGF, TGF, HBEGF,
and epiregulin) originate as transmembrane proteins that are cleaved and
"shed" prior to receptor binding
(1215),
we next sought evidence for the possibility that smoke stimulated the shedding
of ligand into the cell culture medium.
The breast cancer cell line SKBR3 shows EGFR phosphorylation in response to soluble ligand but not to smoke, making the cells suitable "reporters" for smoke-induced ligand shedding. As shown (Fig. 3B), SKBR3 cells displayed strong receptor phosphorylation in response to the conditioned medium from smoke-exposed, but not unexposed, NCIH292 cells. This suggested that NCIH292 cells proteolytically cleaved and released ligand upon smoke exposure and that this ligand was transferred to SKBR cells in the conditioned medium.
The cleavage of EGFR ligands is mediated, at least in some cases, by
transmembrane metalloproteinases called ADAMs
(16,
17). Consistent with the view
that smoke stimulates ligand cleavage and release, we found that the
metalloproteinase inhibitor GM6001 inhibited the phosphorylation of EGFR by
smoke but not EGF (Fig.
4A). When we exposed NCIH292 cells to smoke in the
presence of antisense oligonucleotides corresponding to various ADAMs, we
found that antisense ADAM 17 (tumor necrosis factor converting enzyme)
but not antisense ADAMs 9, 10, or 15 (data not shown for ADAMs 9 and 15)
strongly attenuated smoke-induced phosphorylation of EGFR
(Fig. 4B). Immunoblots
monitoring the amount of ADAM present in the same cells confirmed that
antisense inhibition was appropriate and specific
(Fig. 4C). This
implicated ADAM 17 in the response to smoke contrasting with earlier data
implicating ADAM 10 in the response of the same cells to bacterial LTA
(11).
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Although the basis for substrate specificity of the ADAMs is unclear, it is
recognized that individual ADAMs may cleave more than a single substrate. ADAM
17 in particular has been shown to cleave several surface molecules including
both TNF and TGF
(18). To identify the ligand
cleaved in response to smoke, we prepared immunoblots of medium from
smoke-exposed NCIH292 cells. Although negative for the EGFR ligands
epiregulin, HBEGF, betacellulin, and EGF, the blots showed that smoke but not
LTA strongly stimulated the release of amphiregulin. The reverse was true for
HBEGF (Fig. 4D). Small
amounts of TGF
were also released by smoke (not shown). As expected,
the presence of antisense ADAM 17 inhibited the ability of smoke to stimulate
amphiregulin release (Fig.
4E).
We next addressed the question of how smoke stimulates ADAM 17. Because both EGFR phosphorylation and metalloproteinase activation are known to be redox-sensitive (1923), we asked whether oxygen radicals stimulate ADAM 17 to cleave amphiregulin. As shown (Fig. 5A), smoke exposure raised the levels of ROS in NCIH292 cells. Moreover, antioxidants N-acetyl-L-cysteine (a precursor of glutathione) or dimethylthiourea (an oxygen-radical scavenger) inhibited both ligand release and receptor activation (Fig. 5, B and C).
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Although the appearance of oxygen radicals in nonphagocytic cells is often associated with the diversion of oxygen species from mitochondrial electron transport, recent data show that intracellular radicals may also derive from a low activity NADPH oxidase (24). To examine the relative importance of these two oxidant sources, we exposed cells to smoke in the presence of drugs known to inhibit electron transport (rotenone and antimycin A) or NADPH oxidase (diphenyliodonium chloride). The absence of detectable ROS in diphenyliodonium chloride-treated cells (Fig. 5D) implicated NADPH oxidase in the critical early response to tobacco smoke. Data in Fig. 5 collectively suggest that activation of this oxidase results in amphiregulin cleavage and EGFR phosphorylation, which was shown in Fig. 1 to be necessary for cell proliferation.
To test the relevance of the above findings in an animal model, we exposed mice to smoke in a specially designed chamber (9). Consistent with results in vitro, tracheal lysates from mice exposed to smoke alone showed elevated phosphorylation of EGFR, whereas lysates from animals exposed to smoke in the presence of the metalloproteinase inhibitor GM6001 did not (Fig. 6; compare with data in Fig. 4).
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DISCUSSION |
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These studies also indicate that the ability of EGFR to integrate responses to diverse environmental stimuli depends on stimulus-specific activation of the various ADAMs. When epithelial cells activate EGFR in response to both bacteria (11) and tobacco smoke (this study), they do so by different mechanisms (Fig. 7). In both cases, ADAMs are required, but one involves ADAM 10 and the other ADAM 17. Although the activation mechanisms for these proteases are unknown, our data, obtained in the same cell type using two different stimuli, indicate that these mechanisms are stimulus-specific. Whereas the choice of ADAM itself depends on the nature of the stimulus, the choice of transmembrane ligand would appear to depend upon (a) phenotype-dependent ligand expression and (b) substrate specificity of the activated ADAM. Our data clearly show that both HBEGF and amphiregulin are expressed in NCIH292 cells. Therefore, results showing that LTA elicits cleavage of HBEGF but not amphiregulin (11) and that smoke does the opposite (this study) imply that HBEGF, but not amphiregulin, is a substrate for ADAM 10 and that amphiregulin, but not HBEGF, is a substrate for ADAM 17. Although these findings are in general agreement with evidence linking ADAM 17 to amphiregulin (29) and ADAM 10 to HBEGF (30), they are somewhat at odds with data showing that ADAM 17 is capable of cleaving both amphiregulin and HBEGF (29). Notably, however, the two sets of data were obtained from different cell types (mouse keratinocytes versus human lung epithelial cells). Phenotypic differences with respect to the abundance and location of ADAMs, ligands, and signaling molecules eliciting ADAM activity can be expected to affect the probability of any given cleavage reaction.
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Some disparity can be seen in our own results comparing the degree to which
treatment of cells with antisense ADAM 17 inhibited smoke-induced EGFR
phosphorylation (Fig.
4B, almost complete response) versus ADAM 17
protein levels (Fig.
4C, 50% response). We attribute this to one or both
of the following. 1) Western blot results are not necessarily linear with
protein concentration, and 2) because of the (as yet unknown) subcellular
distribution of ADAM 17, amphiregulin, and EGFR in NCIH292 cells, there may be
a threshold concentration of ADAM 17 below which amphiregulin cleavage is
insufficient to activate EGFR.
In addition to implicating ADAM 17 in the response of lung cells to tobacco smoke, our findings also illustrate that the relevant activation mechanism is oxygen radical-dependent. Oxygen radical production in the setting of smoke exposure appears to be a result of the activation of NADPH oxidase. One possible scenario by which smoke could stimulate this enzyme is suggested by the finding that tobacco smoke induces mitochondrial depolarization (31). This would induce a transient deficiency in ATP production and stimulate compensatory glucose uptake. The conversion of glucose to bisphosphoglycerate would then lead to the production of NADH, which, via the action of a transhydrogenase enzyme, contributes to the formation of NADPH. This serves as a substrate and stimulus for NADPH oxidase.
The uniformity of results obtained in both primary and tumor cells confirms the fundamental nature of the effect of smoke on EGFR and cell proliferation. Although it remains possible that details of the response to smoke differ in normal versus tumor cells, our data suggest that such differences are subtle. This is based on results showing that (a) smoke stimulates proliferation of both non-tumor and tumor cells (Fig. 1, A and B), (b) smoke stimulates EGFR phosphorylation in both cell types (Fig. 2, A and B), (c) there is a causal relationship between smoke-induced EGFR phosphorylation and cell proliferation in both cell types (Fig. 1, A and B), and (d) the mechanism of receptor activation by smoke is metalloproteinase-dependent in both tumor cells in vitro and in mouse tracheal epithelial cells in vivo (Figs. 4 and 6).
In addition to their usefulness as a model, the tumor cell (NCIH292) experiments may directly indicate how smoke stimulates the growth of nascent tumors prior to a diagnosis of lung cancer. An understanding of these signals may suggest strategies to limit the tumorigenic effects of smoke on the lung.
Finally, the phosphorylation of EGFR can be induced by an increasingly long list of environmental stimuli. Because of the role of EGFR in cell proliferation and the relationship between hyperproliferation and malignant transformation, elevated levels of EGFR phosphorylation in a tissue biopsy may identify tissues at risk for malignant transformation. In conjunction with this, it may be possible to identify tumor promoters based on their ability to stimulate the phosphorylation of EGFR in model systems.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Anatomy, University of
California, San Francisco, CA 94143-0452. Tel.: 415-476-3835; Fax:
415-476-4845; E-mail:
cbas{at}itsa.ucsf.edu
1 The abbreviations used are: EGFR, epidermal growth factor receptor; LTA,
lipoteichoic acid; ADAM, a disintegrin and metalloproteinase; ROS, reactive
oxygen species; SFM, serum-free medium; TNF, tumor necrosis factor
; TGF
, transforming growth factor
; HBEGF,
heparin-binding epidermal growth factor; H2DCFDA,
2',7'-dichlorodihydrofluorescein diacetate.
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ACKNOWLEDGMENTS |
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REFERENCES |
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