Departments of 1 Pathology and 2 Pharmacology, University of Vermont College of Medicine, Burlington, Vermont 05405
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We examined the mechanisms of interaction of crocidolite asbestos fibers with the epidermal growth factor (EGF) receptor (EGFR) and the role of the EGFR-extracellular signal-regulated kinase (ERK) signaling pathway in early-response protooncogene (c-fos/c-jun) expression and apoptosis induced by asbestos in rat pleural mesothelial (RPM) cells. Asbestos fibers, but not the nonfibrous analog riebeckite, abolished binding of EGF to the EGFR. This was not due to a direct interaction of fibers with ligand, inasmuch as binding studies using fibers and EGF in the absence of membranes showed that EGF did not adsorb to the surface of asbestos fibers. Exposure of RPM cells to asbestos caused a greater than twofold increase in steady-state message and protein levels of EGFR (P < 0.05). The tyrphostin AG-1478, which inhibits the tyrosine kinase activity of the EGFR, but not the tyrphostin A-10, which does not affect EGFR activity, significantly ameliorated asbestos-induced increases in mRNA levels of c-fos but not of c-jun. Pretreatment of RPM cells with AG-1478 significantly reduced apoptosis in cells exposed to asbestos. Our findings suggest that asbestos-induced binding to EGFR initiates signaling pathways responsible for increased expression of the protooncogene c-fos and the development of apoptosis. The ability to block asbestos-induced elevations in c-fos mRNA levels and apoptosis by small-molecule inhibitors of EGFR phosphorylation may have therapeutic implications in asbestos-related diseases.
mesothelioma; protooncogenes
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ASBESTOS FIBERS, defined as possessing a >3:1 length-to-diameter ratio, are a group of hydrated mineral silicates that can be divided into two families: the serpentine and the amphibole (47). Occupational exposure to asbestos has been linked to the development of malignant (mesothelioma and lung cancer) and nonmalignant (asbestosis) diseases. Crocidolite or "blue" asbestos, an amphibole characterized by a high iron content [Na2Fe3+2(Fe2+,Mg)3Si8O22(OH)2] (47), is the most pathogenic asbestos fiber in the induction of human mesothelioma (30, 31).
Carcinogenic mineral dusts, including crocidolite asbestos, have been
shown to transcriptionally activate a number of early-response genes,
including c-fos,
c-jun, and
c-myc (19, 21, 22, 36, 37, 46). This
gene induction is accompanied by increases in activator protein-1
(AP-1) and activation of nuclear factor-B (21). Nonfibrous analogs
of asbestos and other noncarcinogenic fibers do not elicit these
cellular responses in mesothelial and epithelial cells (22).
The mitogen-activated protein kinase cascade transmits information from extracellular and intracellular stimuli via a series of protein phosphorylation-dephosphorylation events that culminate in the activation of transcription factors and induction of early-response gene transcription. The extracellular signal-regulated protein kinase (ERK) pathway is typically activated at the level of cell surface receptors by mitogens and leads to activation of transcription factors, including that for the c-fos promoter (49). The c-Jun NH2-terminal kinases (JNK/SAPK) are activated by stresses such as irradiation and lead to the activation of c-Jun, a subunit of the AP-1 transcription factor (9). In addition to specific physiological ligands, numerous nonligand agents, such as polycations (29), ultraviolet rays (40, 38), X-irradiation (45), and H2O2 (39, 45), have been shown to interact with and activate growth factor receptors, various signaling moieties, and AP-1 transcription factors. However, little is known about the initiating events leading to asbestos-induced activation of signal transduction pathways and protooncogenes.
Recently, our laboratory has shown that crocidolite asbestos fibers induce activation of the ERK, but not of the JNK/SAPK, cascade in rat pleural mesothelial (RPM) cells (23, 50). Activation of the tyrosine kinase activity of the epidermal growth factor (EGF) receptor (EGFR) is an obligatory step in the asbestos-induced activation of the ERKs (50). The purpose of the present investigation was to assess the effects of asbestos fibers on EGFR binding and biosynthesis. We also wanted to test the hypothesis that activation of the EGFR (and the ERK pathway) by asbestos is causally related to the subsequent activation of early-response genes (i.e., c-fos and c-jun) as well as to apoptosis, an outcome linked to increased early-response gene expression in other cell types (7, 44) and to asbestos exposure in mesothelial cells (1, 16, 23). We show here that crocidolite asbestos, but not its nonfibrous, chemically similar analog riebeckite, interacts with the EGFR and stimulates steady-state mRNA levels and synthesis of EGFR protein. Using a tyrphostin (AG-1478), which specifically blocks autophosphorylation of the EGFR, we also demonstrate that stimulation of EGFR phosphorylation by asbestos is linked to the induction of c-fos, but not of c-jun, by asbestos. Finally, inhibition of signaling through the EGFR-ERK pathway with tyrphostin AG-1478, but not with the nonspecific tyrphostin A-10, decreases apoptosis induced by asbestos in mesothelial cells. These results suggest that pharmacological approaches may be feasible for interruption of asbestos-induced signaling events in progenitor cell types of mesotheliomas.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture and exposure to test agents. RPM cells were isolated from the parietal pleura of Fischer 344 rats by gentle scraping. Cells were propagated in DMEM-F-12 medium (GIBCO BRL, Grand Island, NY) containing 10% fetal bovine serum (FBS), hydrocortisone (100 ng/ml), insulin (2.5 µg/ml), transferrin (2.5 µg/ml), selenium (2.5 ng/ml), penicillin (50 U/ml), and streptomycin (50 µg/ml). When cells were confluent, the growth medium was replaced with medium containing 0.5% FBS for 24 h, then test agents were added for various times. Previously characterized reference samples of National Institute of Environmental Health Sciences-processed crocidolite asbestos fibers from the Thermal Insulation Manufacturers Association fiber repository (Littleton, CO) or riebeckite, a nonfibrous chemically similar analog of crocidolite (29, 49), were sterilized by baking at 250°F overnight and then suspended in Hanks' balanced salt solution (GIBCO BRL) at 1 mg/ml by trituration eight times through a 22-gauge needle before addition directly to medium at final concentrations of 2.5-10.0 µg/cm2 of culture dish. 12-O-tetradecanoylphorbol 13-acetate (TPA; LC Laboratories, San Diego, CA) was dissolved in ethanol and added to medium at a final concentration of 100 ng/ml. EGF (Upstate Biotechnology, Lake Placid, NY) was added to medium at final concentrations of 5-10 ng/ml. Tyrphostin AG-1478 (Calbiochem, San Diego, CA) (26) was dissolved in DMSO and added to medium at 0.1, 1, and 10 µM. Tyrphostin A-10 (LC Laboratories) was also dissolved in DMSO and added at a final concentration of 10 µM (32). In studies using tyrphostins, all dishes, including controls, had a final concentration of 0.1% (vol/vol) DMSO in medium. Cells were pretreated with tyrphostins for 2 h before addition of test agents.
Immunofluorescent EGF binding studies. RPM cells were grown to confluency on glass coverslips, and serum was reduced to 0.5% for 24 h before addition of test agents. Experiments included mock controls and cells preincubated with EGF (5 µg/ml) for 2 h or crocidolite asbestos (5 or 10 µg/cm2) for 2 or 4 h. EGF, biotinylated and complexed to Texas Red streptavidin (Molecular Probes, Eugene, OR), was then added at 500 ng/ml at 4°C for 1 h. Binding of the labeled EGF to the RPM cells was viewed with a fluorescence microscope with use of a wide-band green filter.
Plasma membrane vesicle preparation. All steps were carried out at 4°C. Monolayers of RPM cells were scraped in calcium- and magnesium-free PBS (CMF-PBS). Cells were washed twice with CMF-PBS, then sedimentation was carried out by centrifugation at 400 g. Cells were then resuspended in a lysis buffer [0.25 M sucrose, 2.5 mM magnesium acetate, 25 mM Tris · HCl, pH 7.4, 2.5 mM EGTA, 2 mM dithiothreitol, 1% aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 5 µg/ml leupeptin]. Cells were sonicated on ice and centrifuged at 2,500 rpm at 4°C for 5 min to pellet cell debris and nuclei. The supernatant was then centrifuged at 100,000 g for 1 h at 4°C. The membrane pellet was resuspended in 8.6% (wt/vol) 0.25 M sucrose-5 mM Tris, pH 7.4, by trituration through a 26-gauge needle. Membrane protein was quantitated using the Bio-Rad microassay procedure.
Iodination of EGF. 125I-EGF was prepared by two methods. For the chloramine T method (4), EGF was dissolved in 25 µl of 0.5 M phosphate buffer, pH 7.5, then 1.16 mCi of carrier-free Na125I were dissolved in 10 µl of buffer, and chloramine T (40 µg in 20 µl) was added. The reaction was stopped at 30 s by addition of 100 µg in 100 µl of sodium metabisulfite. An additional 100 µl of 0.1% BSA were added to the solution. The iodinated protein was separated from unreacted Na125I by gel filtration through Sephadex G-50 with a phosphate buffer blocked with 1% BSA. The labeled EGF was stored frozen in the presence of BSA. The specific activity of the EGF at 0.125 ng/µl was 1.68 × 106 cpm/pmol.
125I-EGF was also prepared by the Bolton-Hunter method (2, 24). Five micrograms of human recombinant EGF in 5 µl of 0.1 M borate buffer, pH 8.5, were added to 1.41 mCi of 125I-labeled Bolton-Hunter reagent (DuPont-NEN, Boston, MA), and the reaction mixture was agitated for 15 min at 0°C. Unchanged reagent was reacted with 0.5 ml of 0.2 M glycine in 0.1 M borate buffer, pH 8.5. The reactants were separated by chromatography on a Sephadex G-25 column in 50 mM phosphate buffer containing 0.25% (wt/vol) gelatin and 0.02% sodium azide. The labeled EGF was purified by refiltration using a Sephadex G-50 column in 0.9% NaCl-20 mM HEPES-0.1% BSA. Aliquots from the EGF peak were stored atEGF binding assay. Binding assays (3) were carried out in 96-well plates in a total volume of 250 µl. All plasticware was preblocked with a 1% BSA solution before use. Final reaction concentrations were 20 mM HEPES, pH 7.5, and 0.1% BSA containing 20 µg of membrane preparation in the presence or absence of crocidolite asbestos or riebeckite. The final concentration of asbestos in the binding reactions was calculated to be approximately equivalent to 5 µg/cm2 of cell membrane. An excess of unlabeled EGF (final concentration 4 µM) was added to the tubes in which nonspecific binding was to be measured. Stock solutions were divided into aliquots into microwells containing increasing amounts 125I-EGF to initiate the binding reactions. The binding assay was conducted at room temperature for 1 h. The reaction mixtures were diluted in 1 ml of 20 mM HEPES (pH 7.5) and 0.1% BSA. The dilute reaction mixtures were filtered to separate receptor-bound 125I-EGF from free ligand by use of a vacuum filtration manifold. Reaction mixtures were filtered through glass microfiber filters (Whatman, Maidstone, UK; Schleicher & Schuell, Keene, NH) that had been wetted with HEPES-BSA. After filtration, each filter was washed several times with HEPES-BSA. Filters were then counted in a gamma spectrometer. Samples were analyzed in duplicate. Total binding corresponded to conditions in the absence of unlabeled EGF, across which increasing concentrations of 125I-EGF were titrated. Nonspecific binding corresponded to conditions in which there was an excess of unlabeled EGF. Specific binding was calculated by subtracting nonspecific binding from total binding. All experiments were performed two to three times.
Northern blot analysis.
After experimental treatments, total RNA was extracted from confluent
RPM monolayers by the method of Chomczynski and Sacchi (5). Purity and
concentration were determined by measuring ultraviolet absorbances at
260 and 280 nm. Fifteen micrograms of total RNA were denatured and
fractionated by electrophoresis on a 1.0% agarose-2.25 M formaldehyde
gel, as described previously (43), and hybridized with
-32P-labeled cDNA probes. cDNA
probes were radiolabeled using a random-primer Prime-a-Gene labeling
system (Promega, Madison, WI) (12), then purified over a Sephadex
column before visualization by exposure to Kodak X-OMAT film. The
radioactivity on the blots was directly quantitated using a Bio-Rad
phosphorimager. The rat EGFR cDNA was a gift from Dr. H. Shelton
Earp (University of North Carolina, Chapel Hill, NC). The cDNAs for rat
c-fos and
c-jun were obtained from Dr. Steven
Xanthoudakis (Roche Institute of Molecular Biology, Nutley, NJ) and Dr.
Kazushige Yokoyamu (Riken Gene Bank, Ibaraki, Japan), respectively.
Blots were reprobed, with a rat Cu-Zn superoxide dismutase cDNA probe
(generously provided by Dr. Ye-Shih Ho, Wayne State University,
Detroit, MI) used as a housekeeping probe (12). All experiments were
performed two to three times.
[35S]methionine in vivo labeling of cells and immunoprecipitation of EGFR. To determine degradation of prelabeled EGFR, a method described originally by Earp et al. (11) was used. At confluency, medium (DMEM-F-12, GIBCO BRL, Brewster, NY) was changed to methionine- and cystine-free DMEM for 20 min to deplete intracellular methionine and then supplemented with 5% normal DMEM-F-12 methionine levels, 0.5% FBS, and 50 µCi of [35S]methionine-cysteine (DuPont-NEN) for 18 h. After removal of 35S-containing medium, cells were washed with DMEM-F-12 containing 0.5% FBS, and various test agents were added. Cells then were washed twice with CMF-PBS and scraped into 500 µl of lysis buffer (1% Nonidet P-40, 50 mM Tris · HCl, pH 8.5, 0.15 M NaCl, 5 mM EDTA, 0.1% BSA, 10 µg/ml leupeptin, 25 mM benzamidine, and 1 mM PMSF) before plates were washed with an additional 500 µl of lysis buffer. Lysates then were transferred to microfuge tubes to which 50 µl of 5 M NaCl were added (final NaCl concentration 0.38 M). Cells were disrupted by using a sonicator or by passage through a 26-gauge needle. Cellular debris and nuclei were pelleted by centrifugation at 14,000 rpm for 5 min in a microfuge. Supernatants were transferred to new microfuge tubes and incubated for 30 min at 21°C with an anti-rat EGFR antibody (gift from Dr. John Bergeron, McGill University, Montreal, PQ, Canada). After 10 µl of agarose beads (protein A/G mix) were added to tubes, lysates were incubated for an additional 45 min at 4°C while the tubes were rocked. Immunoprecipitates were pelleted in the microfuge and washed sequentially with 1 ml of the following buffers: 1) 50 mM Tris · HCl, pH 8.5, 0.5 M NaCl, 1 mM EDTA, and 0.5% Nonidet P-40; 2) 50 mM Tris · HCl, pH 8.5, 0.15 M NaCl, 1 mM EDTA, and 0.5% Nonidet P-40; and 3) 10 mM Tris · HCl, pH 8.5, and 0.1% Nonidet P-40. All washes contained 1% aprotinin, 1 mM PMSF, and 25 mM benzamidine. Sixty microliters of a 2× sample buffer were then added to the washed beads, and samples were heated at 98°C for 5 min. Beads were pelleted, and samples matched for total protein content were run on 7.5% SDS-polyacrylamide gels. After they were dry, gels were exposed to Kodak X-OMAT film.
To determine synthesis of nascent EGFR protein, RPM cells were grown in 35-mm six-well plates in DMEM-F-12 containing 10% FBS. At confluency, cells were starved for 24 h in methionine- and cysteine-free DMEM supplemented with 5% normal DMEM-F-12 methionine levels containing 0.5% FBS. After starvation, test agents (EGF or crocidolite asbestos) were added for various times. [35S]methionine-cysteine (50 µCi) was added for the last hour of treatment. Cells were harvested, lysed, immunoprecipitated, analyzed by SDS-PAGE, and quantitated by densitometry as described above. For synthesis and degradation studies, duplicate samples from each group at each time point were evaluated, and experiments were repeated twice. Data from individual experiments were analyzed by ANOVA as described below.ERK activity assay. To determine the specificity of AG-1478 vs. A-10 in modification of ERK activity in EGF-exposed RPM cells, tyrphostins (0.1-10 µM) were added to confluent cells maintained in 0.5% FBS-containing medium for 2 h before addition of 5 ng/ml EGF for 30 min. Cells were then lysed as described previously (50), lysates were centrifuged at 14,000 rpm at 4°C for 10 min, and ERK was immunoprecipitated using an anti-ERK (C-14) antibody reacting primarily with ERK2, but also with ERK1 (Santa Cruz Biotechnologies, Santa Cruz, CA), at a 1:100 dilution. Immunoprecipitates were incubated in a kinase buffer containing 5 µg of myelin basic protein. Incorporation of 32P into substrate was visualized by autoradiography and quantitated on a Bio-Rad phosphorimager.
Assays for apoptosis. After treatment with test agents, confluent RPM cells on glass coverslips were washed with PBS three times for 5 min on ice, then cells were fixed with 4% paraformaldehyde in PBS for 15 min on ice. Paraformaldehyde was neutralized by rinsing dishes with 50 mM NH4Cl for 30 s. Cells were permeabilized with 0.1% Triton X-100 in PBS for 15 min, then subjected to three 5-min washes with PBS. Staining with 4,6-diamidino-2-phenylindole (DAPI) and computer-assisted image analysis for quantitation of apoptotic cells were performed as described previously using the TdT-mediated dUTP nick end-labeling technique to establish criteria for nuclear dimensions (16). Briefly, fixed and permeabilized cells on coverslips (2 per group per time period) were stained with DAPI (0.1 µg/ml), and coverslips were mounted and sealed on glass slides before they were viewed on an Olympus BX-50 immunofluorescence microscope with a wide-band green (Cy3) and a wide-band ultraviolet filter. After images of ~800-1,000 DAPI-stained nuclei were collected, they were evaluated using a statistical model with a <1% chance of misclassification (16). Values are means ± SE expressed as percentage of apoptotic cells in each treatment group.
Statistical analyses. Data from individual experiments were analyzed by ANOVA with use of the Student-Newman-Keuls test for multiple comparisons. All experiments were repeated in duplicate or triplicate.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Crocidolite asbestos prevents binding of EGF to RPM cells.
In Fig. 1, using a
fluorescently tagged EGF, we qualitatively examined the binding of EGF
to RPM cells by immunofluorescent microscopy. Experiments were
performed at 4°C to prevent internalization of the receptor-ligand
complexes. Figure 1A shows the
membrane binding of EGF to RPM cells; this binding can be blocked by an excess of unlabeled EGF (Fig. 1B).
Figure 1, C and
D, shows cells preincubated with
crocidolite for 2 and 4 h, respectively, at 4°C. Under these
circumstances, the binding of EGF to the cells is also blocked, whereas
the nonfibrous particle riebeckite does not have this effect (data not
shown).
|
Crocidolite asbestos, but not riebeckite, abolishes EGF-EGFR
interactions.
To examine EGFR binding in a more quantitative manner, receptor-ligand
binding studies were performed on membrane preparations from RPM cells.
The binding studies in Fig. 2 depict the
specific binding of EGF to RPM membrane preparations in the presence
and absence of particulates. As the concentrations of
125I-EGF added to the membrane
preparations increased, the amount of receptor-ligand complexes also
increased as depicted by bound counts. However, if crocidolite was
incubated with the membrane preparations, this increase in
receptor-ligand binding was not seen (Fig.
2A). In similar experiments to
examine the specificity of the crocidolite effect, receptor binding was
examined in the presence of the nonfibrous riebeckite particle, used
here as a negative control. Unlike crocidolite, riebeckite did not
alter the specific binding of EGF to the membranes compared with ligand binding in the absence of particulate matter (Fig.
2B). To ensure that crocidolite was
not exerting this effect simply by adsorbing the EGF and making it
unavailable for binding, binding studies were performed in the absence
of membrane preparations. In these assays, the specific binding of
125I-EGF to crocidolite asbestos
or riebeckite particles was examined. Figure
2C shows no binding of
125I-EGF to crocidolite asbestos
or riebeckite particles even at high concentrations, suggesting no
effect of crocidolite on receptor binding at the level of ligand
sequestration.
|
EGF and crocidolite asbestos stimulate EGFR synthesis.
After ligand binding and receptor activation, EGFR are internalized and
degraded (11, 41). This then stimulates the cell to increase
transcription and synthesis of the receptor to repopulate the cell
surface. In Figs.
3-5,
we examined the patterns of EGFR degradation and synthesis after
exposure of RPM cells to EGF or asbestos. In comparison to untreated
control cells exhibiting constitutive levels of expression, crocidolite
at 24 h and EGF at 2 and 4 h increased
[35S]methionine
incorporation approximately two- to threefold, respectively, into
immunoprecipitable receptor at 170 kDa (Fig. 3). At earlier time
points, EGF treatment resulted in a time-dependent loss of prelabeled
immunoprecipitable p170 (EGFR) over 0.25, 0.5, 1, and 2 h of EGF
treatment. Prelabeled EGFR was completely degraded by 2 h of EGF
treatment (Fig. 4). In contrast, asbestos exposure for 2 h had no
effect on levels of prelabeled EGFR (Fig. 4). Asbestos exposure for up
to 8 h also had no effects on receptor degradation (Fig. 5).
|
|
|
Asbestos increases steady-state levels of EGFR mRNA.
Figure 6 shows Northern blots of total RNA
hybridized with cDNA complementary to the rat EGFR. In these
experiments we comparatively examined the ability of EGF (5 ng/ml), TPA
(100 ng/ml), or crocidolite asbestos (2.5, 5.0, and 10.0 µg/cm2) to modulate expression
of the EGFR. At 1 h, none of these agents increased EGFR mRNA levels
(data not shown). However, at 4 h, EGF caused an ~3-fold increase and
TPA an ~4.5-fold increase in EGFR message levels. Increases in EGFR
message levels in response to crocidolite asbestos (5 µg/cm2) appeared at 8 h and
were not observed at 10 µg/cm2
asbestos, presumably because this is a cytolytic concentration of
asbestos (22).
|
EGFR signaling partially mediates induction of c-fos by crocidolite.
To examine the role of the EGFR on possible downstream events, patterns
of induction of c-fos and
c-jun mRNA by asbestos in the presence
and absence of tyrphostins were examined at 8 h. We showed previously
that increases in mRNA levels for
c-fos and c-jun are accompanied by elevations in
AP-1 DNA binding activity by asbestos in RPM cells in a dose- and
time-related pattern (19, 22). Typically, these responses appear after
4 h of exposure to asbestos and are increased at 8 and 24 h. In
agreement with earlier findings, exposure to asbestos for 8 h induced
an ~3.5- to 4-fold increase in mRNA levels for
c-fos and ~2-fold increases in mRNA
levels for c-jun (Fig.
7). EGF (5 ng/ml) exposure for 8 h did not
increase c-fos or
c-jun mRNA levels significantly above
baseline values in untreated control groups.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have explored possible interactions of crocidolite asbestos with the EGFR. Moreover, we wanted to determine whether downstream events, i.e., the induction of the protooncogenes c-fos and c-jun, as well as increases in numbers of apoptotic cells by asbestos, were causally related to asbestos-induced phosphorylation of EGFR. Our data suggest that crocidolite exerts its effect on binding at the level of the receptor or the membrane and not at the ligand. Asbestos fibers do not act merely by physically blocking access of the ligand EGF to the cell surface at concentrations of fibers used in these experiments. One reason that receptors fail to bind their ligand is competition; e.g., a saturating amount of unlabeled ligand will occupy receptors, causing them to dimerize and making them unavailable to bind additional labeled ligand molecules. Crocidolite asbestos may interact with EGFR in RPM cell membranes in a manner that promotes receptor dimerization and activation, subsequently preventing receptors from binding native ligand. Although the exact nature of these interactions remains undetermined, it is plausible that physical and chemical effects may occur, i.e., the length of the fiber stretching across the cell membrane (34) in concert with the high iron content of the fibers, which catalytically generates reactive oxygen species intracellularly. For example, agents such as antibodies that cause cross-linking of EGFRs aggregate and activate these receptors and, in turn, inhibit EGF binding (29, 33). In this example, the activation is not directly due to an interaction with the binding site on the receptor kinase but, rather, is aggregation dependent. Various cations have also been shown to activate the EGFR kinase as a direct effect of aggregation (29). These reports indicate that receptor aggregation or dimerization is the causal link between ligand binding to the extracellular domain of tyrosine kinase receptors and the activation of their intracellular kinases and that the same effects can be brought about by nonligand substances. In support of the hypothesis that asbestos fibers cause EGFR aggregation, recent work from our laboratory has shown by immunofluorescence with use of an antibody specific to the external domain of EGFR protein that exposure of human mesothelial cells to long crocidolite fibers leads to aggregation and accumulation of EGFR at sites of fiber contact (34).
Given the finding that asbestos also stimulates biosynthesis of the EGFR and that asbestos activates ERK in an EGFR-dependent manner (50), it was important to determine the role of other elements of the EGFR-ERK pathway in cellular responses to asbestos. A number of molecular responses to asbestos have been characterized in RPM cells, including increased expression of the protooncogenes c-fos and c-jun (19, 22). In support of separate regulatory pathways for these protooncogenes, we recently showed that tyrosine-specific protein kinase inhibitors prevent asbestos-induced c-fos, but not c-jun, expression in RPM cells (14). These results are consistent with data here showing that inhibition of asbestos-induced signaling through the EGFR caused a significant reduction in the induction of c-fos, but not c-jun, mRNA. The lack of complete inhibition of increased steady-state c-fos mRNA levels with use of AG-1478 may reflect the fact that c-fos also is activated by other factors downstream from the EGFR.
Our findings suggest a chain of cell signaling events induced by
asbestos fibers in the mesothelial cell that can be interrupted by
inhibition of EGFR phosphorylation (Fig.
9). Asbestos is not a specific ligand for
the EGFR but may cause activation by enhancing dimerization, possibly
by charge interactions or metal complexes (29). In addition,
crocidolite asbestos generates formation of oxygen free radicals from
redox reactions occurring on the fiber surface (48). Because
phosphorylation of the EGFR by oxidative stress has been demonstrated
in other cell types (39), this may be an alternate mechanism of
activation by asbestos fibers. Exposure to asbestos also causes a
compensatory increase in EGFR steady-state mRNA and protein levels
after activation and internalization of the EGFR that may be
independent of its effects on EGFR phosphorylation.
|
We have shown that, subsequent to EGFR activation, asbestos fibers
cause phosphorylation of Raf-1 and phosphorylation and activation of
ERKs (50). Previous work also shows increases in hydrolysis of membrane
lipids to diacylglycerol by crocidolite asbestos (42), which then
activates protein kinase C (PKC). Although the role of PKC in the
regulation of the ERK pathway remains contentious, asbestos-induced
activation of ERKs is abrogated when PKC is downmodulated by prolonged
treatment of RPM cells with phorbol esters (unpublished data).
Moreover, inhibition of PKC causes a decrease in asbestos-induced
c-fos expression in RPM cells (14).
These data and work showing that PKC- can directly phosphorylate
Raf-1 in vitro (25) suggest that PKC activation is a critical component
of ERK-induced cell signaling by asbestos.
Phosphorylated ERKs translocate to the nucleus to phosphorylate a number of substrates. Transcription factors contributing to the growth factor induction of c-fos are serum response factor (SRF) and ternary complex factor (TCF) or Elk-1. A dimer of SRF and one molecule of TCF bind to the serum response element in the promoter region of c-fos. Others have shown that phosphorylation of SRF and TCF after activation of the ERK pathway appears essential to elicit c-fos transcriptional activation (15, 28). Our model is consistent with these observations, although recent work also shows that phosphorylation of the cAMP response element-binding protein is a prerequisite for c-fos inducibility by EGF (8).
Consistent with Fig. 9, asbestos-induced signaling through the EGFR and subsequent activation of the ERK pathway are linked in our studies to induction of the protooncogene c-fos, as well as the subsequent development of apoptosis in RPM cells. The demonstration that c-fos activation and overexpression of c-Fos protein are causally related to the induction of apoptosis in other cell types (7, 35, 44) is in accordance with our results. Moreover, a recent publication shows that EGFR signaling is necessary for cisplatin-induced apoptosis in tumor cells with use of an antisense approach (10). We recently reported that significant inhibition of apoptosis occurs in RPM cells pretreated with the mitogen-activated protein kinase kinase-1 inhibitor PD-90859, supporting a link between EGFR, ERK activation, and apoptosis (23).
Our findings are novel, in that they show for the first time that interaction of asbestos fibers with a cytokine receptor and subsequent activation of signaling events are linked to a phenotypic end point important in cell injury and repair. Although apoptosis of pulmonary epithelial cells has been implicated in the development of pulmonary fibrosis (17, 18), apoptosis of epithelial or mesothelial cells may also be critical to elimination of initiated cells during the process of carcinogenesis (27).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Douglas Taatjes and S. Martin Shreeve for helpful discussions regarding the imaging and binding studies, respectively. Laurie Sabens helped with manuscript preparation.
![]() |
FOOTNOTES |
---|
This research was supported in part by National Institutes of Health Grants RO1 ES-06499, RO1 ES/HL-09213, RO1 HL-39469, and Environmental Pathology Training Grant T-3207122.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: B. T. Mossman, Dept. of Pathology, University of Vermont, Medical Alumni Bldg., Burlington, VT 05405 (E-mail: bmossman{at}zoo.uvm.edu).
Received 24 December 1998; accepted in final form 15 April 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Berube, K. A.,
T. R. Quinlan,
H. Fung,
J. Magae,
P. Vacek,
D. J. Taatjes,
and
B. T. Mossman.
Apoptosis is observed in mesothelial cells after exposure to crocidolite asbestos.
Am. J. Respir. Cell Mol. Biol.
15:
141-147,
1996[Abstract].
2.
Bolton, A. E.,
and
W. M. Hunter.
The labeling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent.
Biochem. J.
133:
529-539,
1973[Medline].
3.
Carpenter, G.
Binding assays for epidermal growth factor.
Methods Enzymol.
109:
101-110,
1985[Medline].
4.
Carpenter, G.,
and
S. Cohen.
125I-labeled human epidermal growth factor: binding, internalization, and degradation in human fibroblasts.
J. Cell Biol.
71:
159-171,
1976[Abstract].
5.
Chomczynski, P.,
and
N. Sacchi.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal. Biochem.
162:
156-159,
1987[Medline].
6.
Cochet, C.,
G. N. Gill,
J. Meisenhelder,
J. A. Cooper,
and
T. Hunter.
C-kinase phosphorylates the epidermal growth factor receptor and reduces its epidermal growth factor-stimulated tyrosine protein kinase activity.
J. Biol. Chem.
259:
2553-2558,
1984
7.
Day, M. L.,
X. Zhao,
S. Wu,
P. E. Swanson,
and
P. A. Humphrey.
Phorbol ester-induced apoptosis is accompanied by NGFI-A and c-fos activation in androgen-sensitive prostate cancer cells.
Cell Growth Differ.
5:
735-741,
1994[Abstract].
8.
De Cesare, D.,
S. Jacquot,
A. Hanauer,
and
P. Sassone-Corsi.
Rsk-2 activity is necessary for epidermal growth factor-induced phosphorylation of CREB protein and transcription of c-fos gene.
Proc. Natl. Acad. Sci. USA
95:
12202-12207,
1998
9.
Derijard, B.,
M. Hibi,
I. H. Wu,
T. Barrett,
B. Su,
T. Deng,
M. Karin,
and
R. J. Davis.
JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain.
Cell
76:
1025-1037,
1994[Medline].
10.
Dixit, M.,
J. Yang,
M. C. Poirier,
J. O. Price,
P. A. Andrews,
and
C. L. Arteaga.
Abrogation of cisplatin-induced programmed cell death in human breast cancer cells by epidermal growth factor antisense RNA.
J. Natl. Cancer Inst.
89:
365-373,
1997
11.
Earp, H. S.,
K. S. Austin,
J. Blaisdell,
R. A. Rubin,
K. G. Nelson,
L. W. Lee,
and
J. W. Grisham.
Epidermal growth factor (EGF) stimulates EGF receptor synthesis.
J. Biol. Chem.
261:
4777-4780,
1986
12.
Feinberg, A. P.,
and
B. Vogelstein.
A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.
Anal. Biochem.
132:
6-13,
1983[Medline].
13.
Fry, D. W.,
A. J. Kraker,
A. McMichael,
L. A. Ambroso,
J. M. Nelson,
W. R. Leopold,
R. W. Connors,
and
A. J. Bridges.
A specific inhibitor of the epidermal growth factor receptor tyrosine kinase.
Science
265:
1093-1095,
1994[Medline].
14.
Fung, H.,
T. R. Quinlan,
Y. M. W. Janssen,
C. R. Timblin,
J. P. Marsh,
N. H. Heintz,
D. J. Taatjes,
P. Vacek,
S. Jaken,
and
B. T. Mossman.
Inhibition of protein kinase C (PKC) prevents asbestos-induced c-fos and c-jun protooncogene expression in mesothelial cells.
Cancer Res.
57:
3101-3105,
1997[Abstract].
15.
Gille, H.,
A. D. Sharrocks,
and
P. E. Shaw.
Phosphorylation of transcription factor p62TCF by MAP kinase stimulates ternary complex formation at c-fos promoter.
Nature
358:
414-417,
1992[Medline].
16.
Goldberg, J. L.,
C. L. Zanella,
Y. Janssen,
C. R. Timblin,
L. A. Jimenez,
P. Vacek,
D. J. Taatjes,
and
B. T. Mossman.
Novel cell imaging approaches show induction of apoptosis and proliferation in mesothelial cells by asbestos.
Am. J. Respir. Cell Mol. Biol.
17:
265-271,
1997
17.
Hagimoto, N.,
K. Kuwano,
H. Miyazaki,
R. Kunitake,
M. Fujita,
M. Kawasaki,
Y. Kaneko,
and
N. Hara.
Induction of apoptosis and pulmonary fibrosis in mice in response to ligation of Fas antigen.
Am. J. Respir. Cell Mol. Biol.
17:
272-278,
1997
18.
Hagimoto, N.,
K. Kuwano,
Y. Nomoto,
R. Kunitake,
and
N. Hara.
Apoptosis and expression of Fas/Fas ligand mRNA in bleomycin-induced pulmonary fibrosis in mice.
Am. J. Respir. Cell Mol. Biol.
16:
91-101,
1997[Abstract].
19.
Heintz, N. H.,
Y. M. Janssen,
and
B. T. Mossman.
Persistent induction of c-fos and c-jun expression by asbestos.
Proc. Natl. Acad. Sci. USA
90:
3299-3303,
1993[Abstract].
20.
Hunter, T.,
and
J. A. Cooper.
Protein-tyrosine kinases.
Annu. Rev. Biochem.
54:
897-930,
1985[Medline].
21.
Janssen, Y. M.,
A. Barchowsky,
M. Treadwell,
K. E. Driscoll,
and
B. T. Mossman.
Asbestos induces nuclear factor-B (NF-
B) DNA-binding activity and NF-
B-dependent gene expression in tracheal epithelial cells.
Proc. Natl. Acad. Sci. USA
92:
8458-8462,
1995[Abstract].
22.
Janssen, Y. M. W.,
N. H. Heintz,
J. P. Marsh,
P. J. A. Borm,
and
B. T. Mossman.
Induction of c-fos and c-jun protooncogenes in target cells of the lung and pleura by carcinogenic fibers.
Am. J. Respir. Cell Mol. Biol.
11:
522-530,
1994[Abstract].
23.
Jimenez, L. A.,
C. Zanella,
H. Fung,
Y. Janssen,
P. Vacek,
C. Charland,
J. Goldberg,
and
B. T. Mossman.
Role of extracellular signal-regulated protein kinases in apoptosis by asbestos and H2O2.
Am. J. Physiol.
273 (Lung Cell. Mol. Physiol. 17):
L1029-L1035,
1997
24.
Kermode, J. C.,
and
T. R. Tritton.
Receptor-purified, Bolton-Hunter radioiodinated, recombinant, human epidermal growth factor: an improved radioligand for receptor studies.
J. Receptor Res.
9:
429-440,
1989-90.
25.
Kolch, W.,
G. Heidecker,
G. Kochs,
R. Hummel,
H. Vahidi,
H. Mischak,
G. Finkenzeller,
D. Marme,
and
U. R. Rapp.
Protein kinase C activates RAF-1 by direct phosphorylation.
Nature
364:
249-252,
1993[Medline].
26.
Levitzki, A.,
and
A. Gazit.
Tyrosine kinase inhibition: an approach to drug development.
Science
267:
1782-1788,
1995[Medline].
27.
Manning, F. C. R.,
and
S. R. Patierno.
Apoptosis: inhibitor or instigator of carcinogenesis?
Cancer Invest.
14:
455-465,
1996[Medline].
28.
Marais, R.,
J. Wynne,
and
R. Treisman.
The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain.
Cell
73:
381-393,
1993[Medline].
29.
Mohammadi, M.,
A. Honegger,
A. Sorokin,
A. Ullrich,
J. Schlessinger,
and
D. R. Hurwitz.
Aggregation-induced activation of the epidermal growth factor receptor protein tyrosine kinase.
Biochemistry
32:
8742-8748,
1993[Medline].
30.
Mossman, B. T.,
J. Bignon,
M. Corn,
A. Seaton,
and
J. B. L. Gee.
Asbestos: scientific developments and implications for public policy.
Science
247:
294-301,
1990[Medline].
31.
Mossman, B. T.,
and
A. Churg.
State-of-the-art: mechanisms in the pathogenesis of asbestosis and silicosis.
Am. J. Respir. Crit. Care Med.
157:
1666-1680,
1998
32.
Novogrodsky, A.,
A. Vanichkin,
M. Patya,
A. Gazit,
N. Osherov,
and
A. Levitzki.
Prevention of lipopolysaccharide-induced lethal toxicity by tyrosine kinase inhibitors.
Science
264:
1319-1322,
1994[Medline].
33.
Osherov, N.,
and
A. Levitzki.
Epidermal-growth-factor-dependent activation of the Src-family kinases.
Eur. J. Biochem.
225:
1047-1053,
1994[Abstract].
34.
Pache, J. C.,
Y. M. W. Janssen,
E. S. Walsh,
T. R. Quinlan,
C. L. Zanella,
R. B. Low,
D. J. Taatjes,
and
B. T. Mossman.
Increased epidermal growth factor-receptor protein in a human mesothelial cell line in response to long asbestos fibers.
Am. J. Pathol.
152:
333-340,
1998[Abstract].
35.
Preston, G. A.,
T. T. Lyon,
Y. Yin,
J. E. Lang,
G. Solomon,
L. Annab,
D. G. Srinivasan,
D. A. Alcorta,
and
J. C. Barrett.
Induction of apoptosis by c-Fos protein.
Mol. Cell. Biol.
16:
211-218,
1996[Abstract].
36.
Quinlan, T. R.,
K. A. Berube,
J. P. Marsh,
Y. M. W. Janssen,
P. Taishi,
K. O. Leslie,
D. Hemenway,
P. T. O'Shaughnessy,
P. Vacek,
and
B. T. Mossman.
Patterns of inflammation, cell proliferation, and related gene expression in lung after inhalation of chrysotile asbestos.
Am. J. Pathol.
147:
728-739,
1995[Abstract].
37.
Quinlan, T. R.,
J. P. Marsh,
Y. M. W. Janssen,
K. O. Leslie,
D. Hemenway,
P. Vacek,
and
B. T. Mossman.
Dose-responsive increases in pulmonary fibrosis after inhalation of asbestos.
Am. J. Respir. Crit. Care Med.
150:
200-206,
1994[Abstract].
38.
Radler-Pohl, A.,
C. Sachsenmaier,
S. Gebel,
H.-P. Auer,
J. T. Bruder,
U. Rapp,
P. Angel,
H. J. Rahmsdorf,
and
P. Herrlich.
UV-induced activation of AP-1 involves obligatory extranuclear steps including Raf-1 kinase.
EMBO J.
12:
1005-1012,
1993[Abstract].
39.
Rao, G. N.
Hydrogen peroxide induces complex formation of SHC-Grb2-SOS with receptor tyrosine kinase and activates Ras and extracellular signal-regulated protein kinases group of mitogen-activated protein kinases.
Oncogene
13:
713-719,
1996[Medline].
40.
Sachsenmaier, C.,
A. Radler-Pohl,
R. Zinck,
A. Nordheim,
and
P. Herrlich.
Involvement of growth factor receptors in the mammalian UVC response.
Cell
78:
963-972,
1994[Medline].
41.
Schlessinger, J.,
A. B. Schreiber,
T. A. Libermann,
I. Lax,
A. Avivi,
and
Y. Yarden.
Polypeptide-hormone-induced receptor clustering and internalization.
In: Cell Membranes: Methods and Reviews, edited by E. Elson,
W. Frazier,
and L. Glaser. New York: Plenum, 1983, vol. 1, p. 117-148.
42.
Sesko, A.,
M. Cabot,
and
B. T. Mossman.
Hydrolysis of phosphoinositides precedes cellular proliferation in asbestos-stimulated tracheobronchial epithelial cells.
Proc. Natl. Acad. Sci. USA
87:
7385-7389,
1990[Abstract].
43.
Shull, S.,
N. H. Heintz,
M. Periasamy,
M. Manohar,
Y. Janssen,
J. Marsh,
and
B. T. Mossman.
Differential regulation of antioxidant enzymes in response to oxidants.
J. Biol. Chem.
266:
24398-24403,
1991
44.
Smeyne, R. J.,
M. Vendrell,
M. Hayward,
S. J. Baker,
G. G. Miao,
K. Schilling,
L. Robertson,
T. Curran,
and
J. I. Morgan.
Continuous c-fos expression precedes programmed cell death in vivo.
Nature
363:
166-169,
1993[Medline].
45.
Stevenson, M. A.,
S. S. Pollock,
C. N. Coleman,
and
S. K. Calderwood.
X-irradiation, phorbol esters, and H2O2 stimulate mitogen-activated protein kinase activity in NIH-3T3 cells through the formation of reactive oxygen intermediates.
Cancer Res.
54:
12-15,
1994[Abstract].
46.
Timblin, C. R.,
Y. M. W. Janssen,
and
B. T. Mossman.
Transcriptional activation of the proto-oncogene c-jun by asbestos and H2O2 is directly related to increased proliferation and transformation of tracheal epithelial cells.
Cancer Res.
55:
2723-2726,
1995[Abstract].
47.
Veblen, D. R.,
and
A. G. Wylie.
Mineralogy of amphiboles and 1:1 layer silicates.
In: Health Effects of Mineral Dusts, edited by G. D. Guthrie,
and B. T. Mossman. Washington, DC: Mineralogical Society of America, 1993, vol. 28, p. 61-138.
48.
Weitzman, S. A.,
and
P. Graceffa.
Asbestos catalyzes hydroxyl and superoxide radical generation from hydrogen peroxide.
Arch. Biochem. Biophys.
228:
373-376,
1984[Medline].
49.
Whitmarsh, A. J.,
and
R. J. Davis.
Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways.
J. Mol. Med.
74:
589-607,
1996[Medline].
50.
Zanella, C. L.,
J. Posada,
T. R. Tritton,
and
B. T. Mossman.
Asbestos causes stimulation of the extracellular signal-regulated kinase 1 mitogen-activated protein kinase cascade after phosphorylation of the epidermal growth factor receptor.
Cancer Res.
56:
5334-5338,
1996[Abstract].