Activation of the EGF receptor signaling pathway in human airway epithelial cells exposed to metals

Weidong Wu1, Lee M. Graves2, Ilona Jaspers1, Robert B. Devlin3, William Reed1, and James M. Samet3

1 Center for Environmental Medicine and Lung Biology and 2 Department of Pharmacology, University of North Carolina, Chapel Hill 27599; and 3 Human Studies Division, National Health Effects and Environmental Research Laboratory, United States Environmental Protection Agency, Research Triangle Park, North Carolina 27711


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have previously shown that exposure to combustion-derived metals rapidly (within 20 min) activated mitogen-activated protein kinases (MAPK), including extracellular signal-regulated kinase (ERK), in the human bronchial epithelial cell line BEAS. To study the mechanisms responsible for metal-induced activation of ERK, we examined the effect of noncytotoxic exposures to As, Cu, V, or Zn on the kinases upstream of ERK in the epidermal growth factor (EGF) receptor signaling pathway. Western blotting using phospho-specific ERK1/2 antibody demonstrated the selective MEK1/2 inhibitor PD-98059 blocked metal-induced phosphorylation of ERK1/2. Meanwhile, Western blotting using a phospho-specific MEK1/2 antibody showed that these metals induce a rapid phosphorylation of MEK1/2. Kinase activity assays confirmed the activation of MEK1/2 by metal treatment. Immunoprecipitation studies demonstrated that As, Cu, V, or Zn induces EGF receptor phosphorylation. Furthermore, the EGF receptor-specific tyrosine kinase inhibitor (PD-153035) significantly blocked the phosphorylation of MEK1/2 initiated by metals. Interestingly, we observed low levels of Raf-1 activity that were not increased by metal exposure in these cells through kinase activity assay. Finally, transfection assays showed that MEK1/2 inhibition could inhibit trans-activation of Elk1, a transcription factor in the ERK pathway, in BEAS cells exposed to metals. Together, these data demonstrate that As, Cu, V, and Zn can activate the EGF receptor signaling pathway in BEAS cells and suggest that this mechanism may be involved in pulmonary responses to metal inhalation.

signal transduction; air pollution; epidermal growth factor receptor; mitogen-activated protein kinase kinase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MAMMALIAN CELLS RESPOND to extracellular stimuli and stress by activating signaling pathways that lead to cellular responses. Among the major types of signal transduction pathways in eukaryotic cells are protein kinase cascades that culminate in activation of a family of protein kinases known as mitogen-activated protein kinases (MAPK) (10, 22, 29, 32, 56). MAPK are a group of 38- to 110-kDa Ser/Thr kinases that transduce signals that lead to diverse cellular responses (58). Each of the three major MAPK pathways consists of a three-tiered cascade that includes a Ser/Thr MAPK that is phosphorylated by a dual-specificity Thr/Tyr MAPK kinase, MAPKK, which is itself phosphorylated by a Ser/Thr kinase known as a MAPK kinase kinase or MAPKKK (47). Activation of extracellular signal-regulated protein kinases (ERK), one subtype of MAPK, is a key step in the cascade mediating cell proliferation in response to a variety of extracellular signals (9). In contrast, p38 MAPK and p46 to p54 MAPK (Jun NH2-terminal kinases, JNK), two other subtypes of MAPK, mediate signals in response to environmental stress and cytokines. Signaling through the MAPK pathways results in the phosphorylation-dependent activation of a variety of transcription factors (e.g., Elk1, c-Jun, ATF-2) that mediate the specific response to the stimulus (53).

The best-characterized MAPK cascade is the ERK pathway (16). Multiple extracellular stimuli activate the two forms of ERK, also known as p44 and p42. The main pathway leading to ERK activation begins when mitogenic stimuli such as growth factors [e.g., epidermal growth factor (EGF), platelet-derived growth factor] bind to cell surface receptors with intrinsic tyrosine kinase activity (26). This is followed by the recruitment of SH2 domain-containing adaptor proteins (e.g., GRB2 or SHC) to the activated receptors (16). Membrane translocation of the GRB2-SOS complex stimulates Ras-GDP to -GTP exchange, in turn phosphorylating Raf (MAPKKK), MEK (MAPKK), and ERK in series (26). On activation, a portion of the ERK proteins translocates to the nucleus where they phosphorylate and activate at least two transcription factors, c-Myc and Elk1, which mediate changes in gene expression (25).

We have previously reported that acute exposure of the human bronchial epithelial cell line BEAS 2B (BEAS) to As, Cr, Cu, V, or Zn resulted in activation of the ERK, JNK, and p38 MAPK pathways and induced the phosphorylation of the transcription factors c-Jun and ATF-2 in BEAS cells (43). To further characterize the upstream signaling events in ERK activation in human bronchial epithelial cell line BEAS 2B (BEAS) exposed to the combustion-derived metals As, Cu, V, and Zn, this study focused on the activation of MEK, Raf-1, and the EGF receptor tyrosine kinases. We also determined the effect of metal exposure on the transcription factor and ERK substrate Elk1 (3, 39, 53). We report that exposure to As, Cu, V, or Zn activates EGF receptor, MEK1/2, and results in MEK-dependent Elk1 activation in BEAS cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. Tissue culture medium, supplements, and supplies were obtained from Clonetics (San Diego, CA). SDS-PAGE supplies such as molecular-mass standards, polyacrylamide, ready gels, and buffers were obtained from Bio-Rad (Richmond, CA). BSA, 2-mercaptoethanol, and other common laboratory chemicals were purchased from Sigma Chemical (St. Louis, MO). [gamma -32P]ATP (7,000 Ci/mmol) was purchased from New England Nuclear (Wilmington, DE). Protein levels were quantified using a Coomassie blue reagent purchased from Bio-Rad. American Chemical Society-grade metal salts were obtained from Alfa (Ward Hill, MA) or Sigma. Specific anti-phospho MEK1/2 (Ser-217/221) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies were obtained from New England Biolabs (Boston, MA). Protein A-agarose, specific anti-phospho ERK (Tyr204) antibody, anti-Raf-1 polyclonal antibody, agarose-conjugated anti-EGF receptor antibody, p-Tyr HRP antibody, and non-phospho MEK1 and MEK2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). PD-98059 was purchased from Calbiochem-Novabiochem (La Jolla, CA). pFR-Luc and pFA2-Elk1 plasmids were purchased from Stratagene Cloning Systems (La Jolla, CA). The EGF receptor kinase inhibitor PD-153035 (17, 26) was the generous gift from Dr. Shelton H. Earp (Lineberger Comprehension Cancer Center, University of North Carolina at Chapel Hill). The B-raf kinase cascade assay kit was purchased from Upstate Biotechnology (Lake Placid, NY).

Cell culture. BEAS 2B (subclone S6) cells were obtained from Drs. Curtis Harris and John Lechner (National Institutes of Health). The BEAS cell line was derived by transforming human bronchial cells with an adenovirus 12-simian virus 40 (SV40) construct (40). BEAS cells (passages 70-80) were grown to 90-100% confluence on tissue culture-treated Costar 6- or 12-well plates in keratinocyte basal medium (KBM) supplemented with 30 µg/ml bovine pituitary extract, 5 ng/ml human EGF, 500 ng/ml hydrocortisone, 0.1 mM ethanolamine, 0.1 mM phosphoethanolamine, and 5 ng/ml insulin as described previously (36, 45). Cells were placed in KBM (without supplements) for 20-22 h before treatment with metals.

Cell treatment. A suspension of 100 mM sodium arsenite, cupric sulfate, vanadyl sulfate, and zinc sulfate was prepared in water and used as a stock for dilution into KBM at a final concentration of 500 µM (43). PD-98059 stock (50 mM) in DMSO was diluted in KBM to the final 50 µM. BEAS cells were pretreated with PD-98059 for 30 min at 37°C before metal challenges. Cells were then lysed and centrifuged. The supernatants were subjected to SDS-PAGE and Western blotting (43).

Western blotting. BEAS cells treated with metals were lysed in RIPA buffer (1% Nonidet P-40, 0.5% deoxycholate, and 0.1% SDS in PBS, pH 7.4) containing protease inhibitors (1 mM vanadyl sulfate, 0.5 mg/ml aprotinin, 0.5 mg/ml E-64, 0.5 mg/ml pepstatin, 0.5 mg/ml bestatin, 10 mg/ml chymostatin, and 0.1 mg/ml leupeptin). After normalization for protein content, cell extracts were subjected to SDS-PAGE on 11% gradient PAGE gels with a Tris-glycine-SDS buffer (42). Proteins were then electroblotted onto nitrocellulose. The blots were blocked with nonfat milk, washed briefly, and incubated with phospho-specific MEK1/2 or ERK1/2 antibodies in 5% BSA overnight, followed by incubation with HRP-conjugated secondary antibody. Bands were detected using chemiluminescence reagents and film.

Immunoprecipitation. BEAS cells were treated with metals and then lysed with RIPA buffer as above. Cell lysate was immunoprecipitated by incubation with 20 µl of agarose-conjugated anti-EGF receptor antibody for 2 h at 4°C. Immune complexes were collected by centrifugation, washed twice with lysis buffer and once with cold PBS, and finally resuspended in 40 µl of sample loading buffer and boiled for 10 min before being subjected to SDS-PAGE and Western blotting. Tyrosine-phosphorylated EGF receptor was then detected using HRP-conjugated specific anti-phosphotyrosine antibodies (26). Bands were detected using chemiluminescence reagents and film.

Raf-1 kinase activity assay. BEAS cells were lysed with buffer consisting of 20 mM Tris (pH 7.5), 1% Triton X-100, 10% glycerol, 137 mM NaCl, and 2 mM EDTA and containing protease inhibitors 0.25 mM PMSF, 10 nM Microcystin LR, 5 µg/ml leupeptin, and 150 µM Na3VO4. Cell lysates were immunoprecipitated with an anti-Raf-1 antibody (C-12) and protein A-agarose beads. The immune complexes were washed twice with lysis buffer and once with ice-cold PBS. The Raf-1 immune complex kinase activity was measured through a enzyme-coupled assay combining MEK, ERK2, and bovine brain myelin basic protein (MBP) (26). Briefly, immune complexes were incubated with 0.5 µg of recombinant MEK1 and 10 µl of a cold ATP mixture [30 mM beta -glycerophosphate, 60 mM HEPES, pH 7.3, 4 mM EGTA, 1.5 mM dithiothreitol (DTT), 0.45 mM Na3VO4, 30 mM MgCl2, 0.3 mM ATP, and 0.3 mg/ml BSA] for 10 min at 30°C. ERK2 (1.25 µg) was added and incubated for an additional 10 min. Finally, 20 µl of hot ATP mixture (2 µCi [gamma -32P]ATP, 10 µg of MBP, 30 mM beta -glycerophosphate, 60 mM HEPES, pH 7.3, 4 mM EGTA, 1.5 mM DTT, 0.45 mM Na3VO4, 30 mM MgCl2, and 6 µg of BSA) were added and incubated for 10 min at 30°C before stopping the reaction by the addition of 20 µl of 100 mM EDTA (pH 7.5). The reaction mixtures were centrifuged briefly, and 40 µl of each supernatant were spotted onto phosphocellular paper. The papers were then washed six times (5-10 min each) in 10% phosphoric acid, soaked briefly with 100% ethanol, and air-dried before liquid scintillation counting was performed. A-Raf and B-Raf kinase activities were determined using commercially available kits from Upstate Biotechnology.

MEK1 activity assay. The immune complexes were prepared similarly to those for the Raf-1 assay (26). Briefly, cell lysates were incubated with 2 µg of anti-MEK1 antibody for 1.5 h at 4°C. Then 20 µl of protein A-agarose were added, and the mixture was incubated for another 40 min. The immune complexes were washed twice with ice-cold lysis buffer and once with PBS. The MEK1 assay was started by adding 5 µl of ERK2 (250 µg/ml) and 10 µl of cold ATP mixture (30 mM beta -glycerophosphate, 60 mM HEPES, pH 7.3, 4 mM EGTA, 1.5 mM DTT, 0.45 mM Na3VO4, 30 mM MgCl4, 0.3 mM ATP, and 0.3 mg/ml BSA) to immune complexes. After incubation for 10 min at 30°C, 20 µl of hot [gamma -32P]ATP mixture (mentioned above) were added, and the reaction was incubated for an additional 10 min. The reaction was stopped by adding 20 µl of 100 mM EDTA (pH 7.5). The immune complexes were centrifuged at 12,000 g for 1 min, and 40 µl of each supernatant were spotted onto phosphocellular papers. The papers were washed in 10% phosphoric acid six times, rinsed once in 100% ethanol, and air-dried before liquid scintillation counting.

Trans-activation of Elk1. Trans-activation of Elk1 after metal stimulation was analyzed using the PathDetect in vivo signal transduction pathway trans-reporting system (Stratagene) according to the supplied protocol. Briefly, BEAS cells grown to 40-80% confluence in 24-well tissue culture plates were cotransfected with 500 ng of pFR-luc reporter plasmid containing a synthetic promoter with five tandem repeats of the yeast GAL4 binding site that control expression of the Photinus pyralis (American firefly) luciferase gene, 25 ng of pFA2-Elk1, and 25 ng of pSV-beta -galactosidase using 1.5 µg of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) transfection reagent (Boehringer Mannheim). Three to six hours after transfection, cultures were incubated with supplemented KBM for 12 h. BEAS cells were deprived of growth factors in KBM for 24 h and then pretreated with PD-98059 for 1 h. The cells were treated with 500 µM As, Cu, V, or Zn or 100 ng of EGF (as positive control). The medium was removed and replaced with KBM for another 6 h before the cells were lysed as per kit instructions (TROPIX, Bedford, MA). Detection of luciferase and beta -galactosidase was conducted using Dual-Light chemiluminescent reporter gene assay system from TROPIX and an AutoLumat LB953 luminometer (Berthold Analytical Instruments, Nashua, NH). Promoter activity was estimated as specific luciferase activity (luciferase counts per unit beta -galactosidase counts).

Image analysis and statistics. Western blot films were digitized, and band net intensities were quantified using a Millipore Digital Bioimaging System (Bedford, MA). Data are presented as means ± SE. Unpaired Student's t-tests with Bonferroni correction were used for pairwise comparison.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In previous studies, we have observed that exposure of BEAS cells to 500 µM As, Cu, V, or Zn for 15-20 min did not result in significant alterations in cell viability, as assessed by assay of lactate dehydrogenase activity released into the culture medium (6% release) and by microscopic examination (>95% viable) (43).

We have previously shown that under these conditions As, Cu, V, or Zn induced a rapid phosphorylation and activation of MAPK in BEAS cells (ERK1/2). Of the metals, As, V, or Zn induced the most pronounced activation of ERK1/2, with a relatively small activation observed after treatment with Cu or Cr (43). The ERK family of MAPK is activated most potently by mitogenic stimuli such as EGF through phosphorylation by MEK1 or MEK2 (2, 8). To examine the role of MEK1/2 in the activation of ERK1/2 by As, Cu, V, or Zn, BEAS cells were incubated with PD-98059, a selective inhibitor of MEK1/2 activation. As seen previously, Western blotting using phospho-specific ERK1/2 antibodies showed that treatment with As, Cu, V, or Zn for 20 min resulted in phosphorylation of ERK1/2 (42- to 44-kDa bands) (Fig. 1). EGF, used as a positive control, induced the expected marked phosphorylation of ERK1/2. Pretreatment of BEAS with the MEK1/2 inhibitor PD-98059 for 30 min reduced both the metal- and EGF-induced ERK1/2 phosphorylation. PD-98059 alone had no discernible effect on phosphorylation of ERK1/2 (Fig. 1). These data suggested a requirement for MEK1/2 activity in ERK1/2 phosphorylation in BEAS cells exposed to As, Cu, V, or Zn.


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Fig. 1.   Inhibition of PD-98059 on metal-induced phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 in BEAS cells. Confluent BEAS cells were depleted of growth factors for 22 h before stimulation with 500 µM As, Cu, V, or Zn for 20 min. Some cells received 50 µM PD-98059 (PD) or vehicle 30 min before metal treatment [epidermal growth factor (EGF), 100 ng/ml, was given as a positive control]. Twenty minutes after exposure, cells were lysed and protein extracts were subjected to SDS-PAGE followed by Western blotting using a phospho-specific ERK1/2 antibody. Shown are representative bands, with accompanying bars derived from 3 separate experiments. Values are means ± SE; n = 3 separate experiments. Ct, control.

To further investigate the influence of exposure to metals on ERK regulation, MEK1/2 phosphorylation was examined using phospho-specific MEK1/2 antibodies. As shown in Fig. 2, treatment of BEAS with 500 µM As, Cu, V, or Zn or 100 ng/ml EGF for 5 or 20 min induced specific phosphorylation of MEK1/2. EGF and V induced the strongest MEK1/2 phosphorylation at 5 min, whereas the effects of As and Cu were stronger at 20 min of exposure. Zn appeared roughly equivalent in potency at 5 and 20 min.


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Fig. 2.   Phosphorylation of mitogen-activated protein kinase kinase (MEK)-1/2 in BEAS cells exposed to metals. Confluent BEAS cells were deprived of growth factors for 22 h and then exposed to 500 µM As, Cu, V, and Zn for 5 min (top) or 20 min (bottom) (EGF, 100 ng/ml, was given as a positive control). BEAS cells were lysed, and protein extracts were subjected to SDS-PAGE followed by Western blotting using phospho-specific MEK1/2 antibody. Shown are representative results from 3 separate experiments. Nos. are relative optical densities normalized to control value.

To confirm that the increased phosphorylation of MEK1/2 represented MEK1/2 activation, we measured MEK1 kinase activity by immune complex assay using MBP as a substrate. There was a measurable increase in MEK1 activity in BEAS cells exposed to 500 µM As, Cu, V, or Zn or 100 ng/ml EGF. The most pronounced increases were evident with EGF or V at 5 min, with a smaller activation in response to As, V, or Zn after 20 min of exposure. Relatively small increases were seen in MEK1/2 activity in Cu-treated cells (Fig. 3). Similar trends were observed when MEK2 was assayed in the same preparation (data not shown).


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Fig. 3.   Metals activate MEK1 in BEAS cells. After being exposed to 500 µM As, Cu, V, and Zn for 5 or 20 min (EGF, 100 ng/ml, was given as a positive control), BEAS cells were lysed and protein extracts were immunoprecipitated with anti-MEK1 antibody for MEK1 immune complex kinase activity assay with myelin basic protein (MBP) as a substrate. Activity of MEK1 was represented by radioactivity of 32P-labeled MBP. Values are means ± SE; n = 3 separate experiments. * P < 0.05 compared with control.

To ascertain the role of Raf-1 in activation of MEK1/2 in BEAS cells exposed to 500 µM As, Cu, V, or Zn, Raf-1 kinase activity was measured with the use of an immune complex assay. In BEAS cells treated with EGF for 20 min, Raf-1 kinase activity increased nearly threefold. In contrast to the effect of these metals on MEK1 activity, exposure to As, Cu, V, or Zn had no biologically significant effect on Raf-1 activity at 5 or 20 min of exposure, although statistical significance was reached in some instances (Fig. 4). In a separate preparation, we studied the effects of metals on the activities of the other two members of Raf family, A-Raf and B-Raf (34). Similar to the results with Raf-1, no significant increases in the activities of A-Raf and B-Raf were observed at 20 min of exposure to V or Zn or to As or Cu for 5 or 20 min (data not shown). Exposure to V or Zn for 5 min induced small increases in A-Raf activity (1.6- and 1.7-fold, respectively) or B-Raf activity (1.4- and 1.6-fold, respectively), whereas EGF induced only a modest (<2-fold over control) elevation in B-Raf and A-Raf activities at either time point. In addition, pretreatment with 25 µM forskolin (21), an inhibitor of Raf-1 activation, had no significant effect on EGF- or metal-induced MEK1/2 phosphorylation at either 5 or 20 min (data not shown).


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Fig. 4.   Effects of metals on Raf-1 kinase activity in BEAS cells. After being exposed to 500 µM As, Cu, V, or Zn for 5 or 20 min (EGF, 100 ng/ml, was given as a control), BEAS cells were lysed, and protein extracts were immunoprecipitated with Raf-1 antibody against Raf-1. Raf-1 activity was assayed using an immune complex kinase activity assay with MBP as a substrate. Activity of Raf-1 is presented as radioactivity of 32P-labeled MBP. Values are means ± SE; n = 3 separate experiments. Note that error bars are too small to be seen. * P < 0.05 compared with control.

We next investigated the role of EGF receptor phosphorylation on metal-induced activation of MEK1/2. Analysis using specific antibodies to the EGF receptor and phosphorylated tyrosines showed that 500 µM As, Cu, V, or Zn induced EGF receptor tyrosine phosphorylation in BEAS cells at 5 or 20 min of exposure. Among the metals, Zn induced the strongest effects at both time points. V appeared to induce stronger EGF receptor phosphorylation at 5 min than at 20 min, whereas the As and Cu effects required longer incubation times to become apparent. The positive control EGF induced pronounced phosphorylation of the EGF receptor within 5 min that persisted at 20 min (Fig. 5).


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Fig. 5.   Metals phosphorylate EGF receptors (EGFR) in BEAS cells. Cells exposed to 500 µM As, Cu, V, and Zn for 5 min (top) or 20 min (bottom) or 100 ng/ml EGF were lysed and immunoprecipated with agarose-conjugated anti-EGF receptor antibodies. Immunoprecipitates were subjected to SDS-PAGE followed by Western blotting using an anti-phosphotyrosine antibody. Shown are representative results from 3 separate experiments.

To further assess the role of EGF receptor phosphorylation in activation of MEK1/2, we incubated BEAS cells with the EGF receptor tyrosine kinase inhibitor PD-153035 (26). This compound significantly blocked the phosphorylation of MEK1/2 in response to treatment with 100 ng/ml EGF and 500 µM As, Cu, V, or Zn for 20 min (Fig. 6).


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Fig. 6.   Inhibition of PD-153035 metal-induced phosphorylation of MEK1/2 in BEAS cells. Cells depleted of growth factors for 22 h were pretreated with EGF receptor kinase inhibitor (RI) PD-153035 (1 µM, 2 h) and then stimulated with 500 µM As, Cu, V, or Zn, 100 ng/ml EGF, or vehicle (Ct). Cell lysates were subjected to SDS-PAGE and immunoblotted with phospho-specific MEK1/2 antibody. Shown are representative results from 3 separate experiments.

To determine the role of MEK in transcriptional events initiated by metal exposure, we examined the dependence of metal-induced activation of the transcription factor Elk1 on MEK activity in BEAS cells treated with As, Cu, V, or Zn. Elk1 activation was measured in metal-exposed BEAS cells pretreated with the MEK1/2 inhibitor PD-98059 or vehicle using a trans-reporting system consisting of the activation domain of Elk1 fused to the DNA binding domain of GAL4, and a luciferase reporter construct linked to the GAL4 binding site. BEAS cells exposed to 500 µM As, Cu, V, or Zn for 20 min had increased Elk1 activity compared with untreated controls, with the potencies of As, V, and Zn equivalent to that of EGF. Pretreatment of the cells for 1 h with 50 µM PD-98059 ablated the metal-induced and EGF-induced elevations in Elk1-driven reporter activity (Fig. 7).


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Fig. 7.   Inhibition of PD-98059 on metal-induced Elk1 trans-activation in BEAS cells. BEAS cells grown to 40-80% confluence in 24-well tissue culture plates were cotransfected with pFR-luc reporter plasmid, pFA2-Elk1, and pSV-beta -galactosidase using DOTAP transfection reagent. After depletion of growth factors for 24 h, cells were pretreated with PD-98059 for 1 h and then exposed to 500 µM As, Cu, V, or Zn or 100 ng EGF (as positive control). Medium was removed and replaced with keratinocyte basal medium for another 6 h before lysing cells as per kit instructions. Promoter activity was estimated as specific luciferase activity (luciferase counts per unit beta -galactosidase counts); n = 4 separate experiments. * P < 0.05 compared with control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

These studies demonstrate that exposure to noncytotoxic levels of the combustion-derived pollutant metals As, Cu, V, or Zn can induce EGF receptor phosphorylation and MEK1/2 activation and link these events to the phosphorylation of ERK1/2 and activation of Elk1 in BEAS cells. These findings show that exposure to the air pollutant metal As, Cu, V, or Zn initiates activation of the EGF receptor signaling cascade in human airway epithelial cells.

MEK1/2 acts as a dual-specificity MAPK kinase in the ERK pathway and is the only identified activator of ERK1/2. Our finding that As, Cu, V, or Zn exposure resulted in ERK1/2 phosphorylation in a MEK1/2-dependent manner in BEAS cells was consistent with the expected role of MEK1/2 in this pathway. Previous studies have shown that sodium arsenite activates MAPK, including ERK, in different cell types but did not study its effect on MEK1/2 activity (1, 11, 28, 31, 54). MEK1/2 activation is the result of specific phosphorylation of the catalytic domains by upstream protein kinases (13, 24). Using phospho-specific MEK1/2 antibodies, we detected phosphorylation of MEK1/2 in BEAS cells treated by As, Cu, V, or Zn. Activation was confirmed by MEK1 activity assays, which indicated an increased activity of MEK1 induced by the metals.

What potential mechanisms can account for the activation of MEK1/2 triggered by As, Cu, V, or Zn? One possibility for activation of MEK1/2 by these metals may be the result of the inhibition of upstream protein tyrosine phosphatases. Although the MAPKK MEK1/2 is itself phosphorylated on serine residues and, therefore, dephosphorylated by a serine phosphatase, the MAPKKK are phosphorylated on tyrosine residues (47). All known protein tyrosine phosphatases (PTPases), including the dual-specificity enzymes that dephosphorylate MAPK, are SH enzymes and sensitive to thiol reagents (52). Some metals, for example, As and Zn, have a high affinity for SH groups. As3+ reacts most readily with vicinal thiols and to a lesser degree thiols proximal to hydroxyl-bearing residues (7). There is evidence that As activates JNK and p38 in Hela cells through inhibiting a dual-specificity Thr/Tyr phosphatase (11). V can activate MAPK in a variety of cell types, possibly through its potent inhibition of PTPase activity (18, 19, 38, 59). Recently, we observed that As, Cu, V, and Zn ions induce a persistent accumulation of PTPases (46) and that Cu, V, and Zn inhibit tyrosine phosphatases in BEAS cells (J. M. Samet, R. Silbajoris, W. Wu, and L. M. Graves, unpublished data). Zn has also been shown to inhibit several PTPases, including the receptor tyrosine phosphatase HPTP-beta (55). In addition, the transition metals Cu and V support redox cycling and generate reactive oxygen species such as H2O2, which is also a potent PTPase inhibitor (20, 50). It is therefore possible that Cu, V, or Zn exposure activates MEK1/2 by inhibiting a phosphatase that dephosphorylates MEK1/2, allowing the phosphorylated form to accumulate above threshold levels needed to effect ERK1/2 activation.

Another possible mechanism for metal-induced activation of MEK is cross talk between the three major branches of the MAPK cascade or other signaling pathways (23, 51). MEKK1,2,3, which functions as a MAPKKK in JNK pathway, can also activate MEK1/2 (6, 33). In addition, protein kinase C-zeta is reported to be capable of phosphorylating MEK kinase, with ensuing activation of the ERK pathway in response to ANG II (27).

Not surprisingly, the effects of As, Cu, V, and Zn exposure on constituents of the EGF receptor cascade displayed some variability. For instance, V induced activation of MEK1/2 earlier than did As, Cu, or Zn exposure. Cu was consistently weaker than the other metals as an activator of EGF receptor-mediated signaling. In addition, some disparities were observed between the phosphorylation and activation of MEK1/2 induced by Cu or Zn. For instance, As and Cu induced a weaker MEK1 activity increase than the phosphorylation of MEK1/2 would have predicted. These dissociations may conceivably be due to hyperphosphorylation-mediated inhibition of MEK1/2 activity, different kinetics between phosphorylation and activation, or a lack of necessary cofactors in the in vitro assay (43). We have recently obtained data suggesting that As, Cu, V, or Zn exposure can activate ras in BEAS cells with varying effectiveness (Wu, Graves, Devlin, and Samet, unpublished data). It will be interesting to see whether the ability to activate Ras correlates with the activation of MAPK members by these metals.

The activation of MEK1/2 is preceded by the activation of an upstream MAPKKK, generally believed to be Raf-1. Activated Raf-1 then phosphorylates MEK on the conserved serine residues within kinase subdomain VIII, thereby activating it (16). However, in this study, we did not observe any clear evidence of Raf-1 activation upon exposure of BEAS cells to As, Cu, V, or Zn and only a modest activation in response to EGF, although a slight increase in A-Raf or B-Raf activity in BEAS cells exposed to V or Zn at 5 min was observed. Although we cannot completely rule out a methodological shortcoming that explains our inability to show metal-induced Raf-1 activation, the fact that the Raf-1 inhibitor forskolin (32) had no discernible effect on MEK1/2 phosphorylation supports a mechanism for MEK1/2 that is Raf-1 independent in metal-treated BEAS cells. Precedence for Raf-1-independent phosphorylation of MEK1/2 exists in published reports from studies in other cell types. ANG II stimulates ERK and JNK activity through a putative Ras/Raf-independent pathway in GN4 rat liver epithelial cells (26). Activation of MEK independently of Raf-1 has also been reported by Winston et al. (57), who found that exposure of mouse macrophages to tumor necrosis factor-alpha stimulated a time-dependent increase in the activity of MEK, with no activation of either c-Raf-1 or Raf-B.

Induction of EGF receptor tyrosine phosphorylation by agents other than EGF family member ligands has been reported previously (26, 41, 60). Our data showing that As, Cu, V, or Zn exposure can activate EGF receptors in BEAS cells are similar to previous findings by Chen et al. (12) who observed the role of EGF receptor in mediating arsenite-induced protein tyrosine phosphorylation in PC-12 cells. Although the mechanism responsible for metal-induced activation of EGF receptor tyrosine kinase is unknown, it is possible to speculate that these metals may directly interact with the EGF receptor molecules, causing a structural alteration or dimerization that results in activation of the kinase domain. For example, the sulfhydryl-reactive metals As and Zn may interact with SH groups on the EGF receptor in a manner that causes its activation. As discussed above, it is also possible that the metals directly or indirectly inactivate phosphatases that serve to dephosphorylate the EGF receptor, allowing the phosphorylated form to accumulate. Another potential mechanism responsible for metal-induced activation of EGF receptor tyrosine kinase may involve the generation of reactive oxidative species in BEAS cells that could lead to membrane changes such as lipid peroxidation products and membrane polarization (49).

Epithelial cells lining the respiratory tract interact with combustion particles or soluble components derived from inhaled combustion by-products, including As, Cu, V, or Zn, which effect inflammatory responses in the lung. The BEAS 2B cell line used in this study is an SV40-transformed human bronchial epithelial cell line, which has been shown to be an excellent surrogate for primary human airway epithelial cells (14, 30, 37, 44, 45). We have previously observed that As, Cu, V, or Zn exposure induces increased release of the inflammatory cytokines interleukin-6, interleukin-8, and tumor necrosis factor-alpha , which has been shown to be regulated through signaling pathways that involve MAPK and activation of MAPK-dependent transcription factors (4, 5, 15, 35, 48). These transcription factors play critical roles in the induction of immediate-early genes and in the mitogenic response (16). Thus our finding that As, Cu, V, and Zn increased Elk1 trans-activation in BEAS in a MEK1/2-dependent manner, together with the activation of EGF receptor tyrosine kinase, implicates activation of this signaling pathway as a mechanism that mediates transcriptional events that underlie pulmonary responses to metal inhalation.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the technical assistance of Yaqin He and Lisa Dailey.


    FOOTNOTES

This work was supported by Environmental Protection Agency Grant CR 817643.

The research described in this article has been reviewed by the Health Effects and Environmental Research Laboratory, United States Environmental Protection Agency, and has been approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of tradenames constitute endorsement or recommendation for use.

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: J. M. Samet, EPA Human Studies Facility CB no. 7315, 104 Mason Farm Rd., Chapel Hill, NC 27599-7315 (E-mail: samet.jim{at}epamail.epa.gov).

Received 24 February 1999; accepted in final form 14 July 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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