1 Center for Environmental
Medicine and Lung Biology and
2 Department of Pharmacology, We have previously
shown that in vitro exposure to metallic compounds enhances expression
of interleukin (IL)-6, IL-8, and tumor necrosis factor-
signal transduction; airway epithelial cells; metal toxicology; air
pollution; mitogen-activated protein kinases
HUMAN EXPOSURE TO AIRBORNE metals induces pulmonary
inflammatory responses, such as tracheobronchitis, asthma, chemical
pneumonitis, and alveolitis (metal fume fever; see Ref. 38), which have
been associated with elevated levels of the cytokines interleukin
(IL)-8, IL-6, and tumor necrosis factor- The intracellular signaling mechanisms responsible for the enhanced
expression of inflammatory proteins by human airway epithelial cells
exposed to metals have not been fully elucidated. However, we recently
reported that exposure of a bronchial epithelial cell line to
V-containing metallic particulate matter results in a rapid and
persistent accumulation of Tyr phosphorylated proteins (50). This
suggested a role for phosphorylation-dependent signaling pathways in
inducing enhanced expression of inflammatory proteins by metallic
compounds. Moreover, acute exposure to V (21, 41, 68), As (5), Cr (26),
or Zn (20) at concentrations ranging from 300 to 500 µM has been
shown to activate components of the phosphorylation-dependent
mitogen-activated protein kinase (MAPK) signaling cascades in various
cell types.
The MAPK pathways transduce signals that lead to diverse cellular
responses, such as cell growth, differentiation, proliferation, apoptosis, and stress responses to environmental stimuli (for reviews
see Refs. 16, 28, 33, 36, 53, 63, 67). 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,
which is, in turn, phosphorylated by a MAPK kinase kinase. The
extracellular receptor kinase (ERK) pathway typically transduces growth
factor signals that lead to cell differentiation or proliferation (33),
whereas cytokines and stress signals (e.g., ultraviolet irradiation,
heat, synthesis inhibitors) activate the c-Jun
NH2-terminal kinase (JNK) and P38
pathways, resulting in stress responses, growth arrest, or apoptosis
(2, 30, 45, 63, 67). Signaling through the MAPK pathways culminates in
the phosphorylation-dependent activation of a variety of transcription factors that modulate cytokine gene expression (53, 57, 65).
The transcription factor c-Jun is a major phosphorylation target of JNK
(7). c-Jun is a component of the activator protein-1 (AP-1) heterodimer
that binds to the TRE/AP-1 DNA response element and regulates IL-6 and
IL-8 gene expression (37). Similarly, ATF-2, a substrate for P38 and
JNK, binds to AP-1 and CRE response elements and is involved in the
expression of TNF- To characterize signaling pathways activated by metals in human
bronchial epithelial cells and to identify metals that activate them,
we examined the effects of soluble forms of the environmentally relevant metals As, Cr, Cu, Fe, Ni, V, and Zn on MAPK signaling, transcription factor activation, and IL-8 expression in the human bronchial epithelial cell line BEAS 2B (BEAS). We report here that
acute exposure to As, Cr, Cu, V, or Zn results in activation of the
ERK, JNK, and P38 MAPK pathways and induces the phosphorylation of the
transcription factors c-Jun and ATF-2 in BEAS cells.
Reagents. Tissue culture medium,
supplements, and supplies were obtained from Clonetics (San Diego, CA).
SDS-PAGE supplies such as molecular-mass standards, polyacrylamide, and
buffers were obtained from Bio-Rad (Richmond, CA). Bovine serum albumin (BSA), 2-mercaptoethanol, phorbol 12-myristate 13-acetate (PMA), and
other common laboratory chemicals were purchased from Sigma Chemical
(St. Louis, MO).
[ Tissue 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 epithelial cells with an ad12-SV40 adenovirus construct (46). BEAS cells were grown to 80-90% confluence on tissue
culture-treated plastic dishes in keratinocyte basal medium (KBM)
supplemented with 30 µg/ml bovine pituitary extract, 5 ng/ml human
epidermal growth factor, 500 ng/ml hydrocortisone, 0.1 mM ethanolamine, 0.1 mM phosphoethanolamine, and 5 ng/ml insulin as described previously (39, 49). BEAS cells that had been passaged 80-100 times in our
laboratory were used for the present studies. Cells were placed in KBM
(without supplements) for 8-14 h before each experiment. For the
IL-8 determinations, the cells were treated with vehicle (KBM alone),
500 µM As, Cr III, Cu, V, or Zn for 20 min, the medium was then
removed, the cells were washed in Hanks' buffered saline, and fresh
KBM was added. The medium was then sampled at 6 and 24 h and kept
frozen Western blotting. Levels of
phosphorylated JNK, ERK1/2, P38, ATF-2, and c-Jun in control and
metal-treated cells were determined by SDS-PAGE as described previously
(47) using phosphospecific antibodies. Briefly, proteins from adherent
cells were extracted with a lysis buffer consisting of 100 mM
Tris · HCl, pH 7.5, 0.1 M NaCl, 2 mM EDTA, and 1%
Nonidet P-40, containing 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml aprotonin, and 1 µg/ml leupeptin, for 10 min
while on ice. Protein extracts were mixed with an equal volume of 0.125 M Tris, pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, and
0.05% bromphenol blue, boiled, and run on 7.5 or 12% SDS-PAGE gels
(31). Prestained molecular-mass markers (Sigma) were run on adjacent
lanes. Samples were normalized for protein content before loading.
Electrophoresed proteins were electroblotted onto nitrocellulose (56),
and the blots were blocked with 5% casein, washed briefly, and
incubated overnight with the primary antibody in 3% BSA.
HRP-conjugated goat anti-rabbit antibody was used as a secondary
antibody. Bands were detected using chemiluminescence reagents and film
as per the manufacturer's instructions (New England Biolabs).
In-gel kinase activity assay. Protein
kinase activities in cell lysates fractionated by SDS-PAGE were
measured as described by Wang and Erikson (60). Briefly, cells were
lysed in a low-salt buffer containing 1% Triton X-100, 25 mM Tris, pH
7.5, 2 mM EGTA, 10% glycerol, 1 mM PMSF, 1 mM sodium metavanadate, 10 mM sodium fluoride, 1 µg/ml pepstatin, and 1 µg/ml leupeptin.
Lysates were loaded onto standard 11% SDS-polyacrylamide gels
containing 250 µg/ml MBP, which was added to the gel just before
polymerization. Samples were normalized for protein content before
loading 50-100 µg of sample protein per well. After running, the
gels were washed sequentially with 20% 2-propanol-50 mM Tris (pH 8.0),
50 mM Tris (pH 8.0)-0.05% 2-mercaptoethanol (buffer
A), and 6 M guanidine hydrochloride in
buffer A (two 30-min washes in each
solution), followed by repeated washings in 0.04% Tween in
buffer A overnight at 4°C. The
phosphorylation reaction was carried out by adding 10 ml of 40 mM HEPES
(pH 8), 2 mM dithiothreitol (DTT), 100 µM EGTA, 5 mM
MgCl2, 25 µM ATP, and 250 µCi
[ MAPK activity assays. For the JNK,
ERK2, and P38 activity assays, cells were lysed in a low-salt buffer
containing 150 mM NaCl, 20 mM Tris · HCl, pH 7.5, 5 mM EDTA, 1% Triton X-100, 50 mM NaF, 10% glycerol, 1 mM sodium
metavanadate, 4 µg/ml aprotonin, 20 µg/ml PMSF, 10 µg/ml
leupeptin, and 1 µg/ml microcystin, and ERK2 or P38 proteins were
immunoprecipitated from 50-100 µg of cell lysate using
agarose-conjugated anti-ERK2 or anti-P38 antibodies for 2 h at 4°C.
The immunoprecipitates were then washed first with lysis buffer and
then with a kinase buffer consisting of 20 mM HEPES, pH 7.4, 10 mM
MgCl2, 50 mM NaCl, 1 mM DTT, 5 mM
B-glycerophosphate, 1 µg/ml microcystin, and 1.5 mM EGTA. Kinase
activity in the immunoprecipitates was measured in kinase buffer
containing 50 µM ATP, 10 µCi
[ Image analysis and statistics. Western
blot films were digitized, and band optical densities were quantified
using a Millipore Digital Bioimaging System (Bedford, MA). In-gel
kinase activities were quantified with a Molecular Dynamics 400E
PhosphorImager radioactivity detector (Sunnyvale, CA). Data are
presented as means ± SE. IL-8 data comparisons were carried out
using one-way ANOVA followed by Dunnett's post hoc test for multigroup
comparisons.
Previous studies have demonstrated activation of MAPKs in several cell
types using concentrations of As, Cr, V, and Zn in the range of
300-500 µM (5, 20, 21, 26, 41, 68). We therefore determined
whether short-term exposure to these concentrations of sodium arsenite
(As), Cr III, potassium perchromate (Cr VI), cupric sulfate (Cu),
vanadyl sulfate (V), or Zn could result in overt cytotoxicity in BEAS
cells. Treatment with 500 µM of these metals for 15-20 min did
not result in significant alterations in BEAS cell viability, as
assessed by assay of lactate dehydrogenase activity released into the
culture medium ( To survey the effect of acute metal exposure on kinase activity in BEAS
cells, we used an in-gel activity assay to measure kinase activities in
BEAS cells exposed to vehicle or 500 µM As, Cr III, Cr VI, Cu, V, or
Zn. Cell extracts from untreated BEAS cells showed a number of bands
corresponding to protein kinase activities of molecular masses ranging
from 40 to 110 kDa (Fig. 1). Relative to
unstimulated cells, treatment of BEAS cells with As, Cr, Cr, Cu, V, or
Zn or with 100 nM PMA for 20 min resulted in a differential activation
of kinases of varying molecular masses (Fig. 1). The strongest and most
consistent increases in activity were evident as dark bands at
~55-60 kDa in cells treated with As and in the range of
40-45 kDa in cells treated with V, Zn, and PMA. Less intense
activation was seen in the 40- to 45-kDa band in cells exposed to As,
Cr III, Cr VI, Cu, and Ni. Exposure to Fe appeared to have no effect on
kinase activity relative to control levels (Fig. 1).
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
in human
bronchial epithelial cells. To characterize signaling pathways involved
in metal-induced expression of inflammatory mediators and to identify
metals that activate them, we studied the effects of As, Cr, Cu, Fe,
Ni, V, and Zn on the mitogen-activated protein kinases (MAPK)
extracellular receptor kinase (ERK), c-Jun
NH2-terminal kinase (JNK), and P38 in BEAS cells. Noncytotoxic concentrations of As, V, and Zn induced a
rapid phosphorylation of MAPK in BEAS cells. Activity assays confirmed
marked activation of ERK, JNK, and P38 in BEAS cells exposed to As, V,
and Zn. Cr and Cu exposure resulted in a relatively small activation of
MAPK, whereas Fe and Ni did not activate MAPK under these conditions.
Similarly, the transcription factors c-Jun and ATF-2, substrates of JNK
and P38, respectively, were markedly phosphorylated in BEAS cells
treated with As, Cr, Cu, V, and Zn. The same acute exposure to As, V,
or Zn that activated MAPK was sufficient to induce a subsequent
increase in IL-8 protein expression in BEAS cells. These data suggest
that MAPK may mediate metal-induced expression of inflammatory proteins
in human bronchial epithelial cells.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
(TNF-
; see Refs. 3 and
29). Similarly, recent studies have shown that acute exposure of rats to mixtures of metallic compounds derived from ambient air particulate matter or from combustion source emissions (e.g., residual oil fly ash)
can induce pronounced pulmonary inflammation characterized by increased
permeability to protein and neutrophilic alveolitis (12, 27, 42, 43).
These inflammatory changes are accompanied by increased synthesis of
the cytokines IL-1, IL-5, and IL-6 (27). Corroborating these clinical
observations and animal studies, in vitro studies have reported
increased synthesis of IL-6 and IL-8 by human airway epithelial cells
exposed to metallic mixtures derived from combustion processes (4, 44).
Moreover, the transition metals V, Cr, and Zn have been shown to induce
cytokine expression in various cell types (13, 52, 61).
(58). Similar to the MAPK pathways, the
transcriptional activities of c-Jun and ATF-2 are induced by a
broad range of stress signals, such as genotoxic agents,
cytokines, and ultraviolet irradiation (23, 35, 66).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-32P]ATP (7,000 Ci/mmol) was purchased from NEN (Wilmington, DE). Protein levels were
quantified using a Coomassie blue reagent purchased from Bio-Rad. Stock
solutions (100 mM) of cupric sulfate, ferrous sulfate, nickel sulfate,
vanadyl sulfate, zinc sulfate, potassium perchromate (Cr VI), chromium
sulfate (Cr III), and sodium arsenite were prepared in water and kept
frozen until ready for use. American Chemical Society-grade metal salts
were obtained from Alfa (Ward Hill, MA) or Sigma. Specific
anti-phospho-JNK (Thr183/Tyr185),
anti-phospho-ERK (Tyr204),
anti-phospho-P38
(Thr180/Tyr182),
anti-phospho-Elk1 (Ser383),
anti-phospho-ATF-2 (Thr71),
anti-phospho-c-Jun (Ser73), and
horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary
antibodies were obtained from New England Biolabs (Boston, MA) and used
as instructed by the manufacturer. Human IL-8 ELISA kits were purchased
from Endogen (Cambridge, MA). Agarose-conjugated anti-ERK2 and anti-P38
antibodies and recombinant full-length human ATF-2 were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA). Bovine brain myelin basic
protein (MBP) was purified as described previously (8). GST-c-Jun
fusion protein was prepared as described previously (69).
80°C until analyzed for IL-8 protein levels.
-32P]ATP for 60 min at room temperature. The gel was then washed extensively overnight
with 5% TCA-1% sodium pyrophosphate, dried, and exposed to film.
-32P]ATP, and
either 25 µg/ml MBP (for ERK samples) or 1 µg ATF-2 (for P38
samples). The same conditions were used for the JNK activity assay
except that JNK protein was precipitated by incubating the cell lysates
with agarose-conjugated GST-c-Jun, the enzyme substrate complex was
then washed, and the reaction was initiated by adding the same kinase
mixture used for the ERK and P38 assays. Kinase reactions were allowed
to proceed for 20 min at 30°C with vigorous agitation in a
Eppendorf ThermoMixer (Brinkman Instruments, Westbury, NY) and were
terminated by adding loading buffer and heating to 95°C for 5 min.
Samples were then subjected to SDS-PAGE, the gels were dried, and the
bands were detected by autoradiography.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
6% release) and by microscopic examination (>95%
viable).
View larger version (88K):
[in a new window]
Fig. 1.
In-gel kinase activity assay of BEAS cells treated with metals. BEAS
cells were incubated for 15 min with medium alone (Ct), with 500 µM
sodium arsenite (As), chromium trioxide (Cr III), potassium perchromate
(Cr VI), cupric sulfate (Cu), ferrous sulfate (Fe), nickel sulfate
(Ni), vanadyl sulfate (V), or zinc sulfate (Zn), or with 100 nM phorbol
12-myristate 13-acetate (PMA). The cells were then lysed, and extracted
proteins were subjected to SDS-PAGE on polyacrylamide gels impregnated
with myelin basic protein. After electrophoresis, the proteins were
renatured, and intrinsic kinase activities were assayed by incubating
the gel with
[ -32P]ATP. Bands
were visualized by autoradiography. See MATERIALS AND
METHODS for additional details.
The apparent molecular masses of the kinases activated by metal treatment of BEAS were consistent with that of the ERK1/2 (42-44 kDa), JNK (55 kDa), and, possibly, P38 (38 kDa) MAPKs. To determine whether exposure to metals ions can induce the activation of MAPKs, protein extracts from BEAS cells treated with 500 µM As, Cr III, Cr VI, Cu, Fe, Ni, V, or Zn ions or 100 nM PMA were subjected to SDS-PAGE followed by Western blotting using phosphospecific anti-ERK1/2, anti-JNK, and anti-P38 antibodies. As suggested by the in-gel kinase activity results, As, Cr III, Cr VI, V, Zn, and PMA induced phosphorylation of the MAPKs ERK2, JNK, and P38 (Fig. 2). Relative to the other metals, V was a potent activator of all three MAPKs. As, Cr III, Cr VI, Cu, and Zn were roughly equipotent in inducing phosphorylation of ERK2 (Fig. 2A). Because the phosphospecific antibody used for the ERK1/2 blot preferentially recognizes phospho-ERK2, an assessment of the effect of metal exposure on ERK1 phosphorylation was not possible. Zn appeared equivalent to V as a stimulus of JNK phosphorylation, whereas As, Cr III, Cr VI, Cu, and Ni had weaker effects (Fig. 2B). In the case of P38, As, Cr III, Cr VI, and Zn were approximately as effective as V in inducing strong phosphorylation relative to unexposed cells (Fig. 2C). As predicted by the in-gel kinase data, treatment with Fe did not result in phosphorylation of MAPKs in BEAS cells (Fig. 2). Exposure to Ni produced generally weak and variable effects on MAPK phosphorylation (Fig. 2). PMA, used as a positive control in these studies, produced the expected stimulation of MAPK phosphorylation in BEAS cells (Fig. 2). Because Cr III and Cr VI appeared roughly equivalent in potency (Figs. 1 and 2), only Cr III, the predominant species in ambient air (24), was used in subsequent experiments.
|
To obtain a direct measure of the effect of the active metals on MAPK activities in bronchial epithelial cells, we measured the effect of BEAS cell exposure to 500 µM As, Cr, Cu, V, or Zn on immunoprecipitated ERK2, JNK, and P38 activities using exogenous substrates. As shown in Fig. 3 and in agreement with the in-gel kinase and Western blot data (Figs. 1 and 2), V was a potent activator of ERK2, JNK, and P38 activities (17.4 ± 3.8-, 9.1 ± 4.2-, and 3.3 ± 0.6-fold over controls, respectively). Similarly, As was an effective stimulus that induced the activation of all three MAPKs while producing the strongest effects on JNK (16.5 ± 4.1-fold increase) and P38 activities (3.9 ± 0.3-fold increase; Fig. 3). Exposure to Zn induced a clear increase in ERK2 activity (7.6 ± 3.3-fold) and a modest elevation in P38 activity (1.7 ± 0.3-fold; Fig. 3, A and C). In contradiction with the strong phosphorylation of JNK observed in the Western blots (Fig. 2B), the effect of Zn on JNK activity was only a modest (2.4 ± 1.1-fold) increase compared with the strong activation induced by As and V (Fig. 3B). As suggested by the in-gel kinase and Western blot findings (Figs. 1 and 2), treatment of BEAS cells with Cr or Cu induced minor activation of ERK2 and P38 relative to the effects induced by V exposure (Fig. 3).
|
To assess the potential effect of MAPK-mediated signaling on gene expression in human bronchial epithelial cells exposed to metals, we next determined the effect of treatment with As, Cr, Cu, V, or Zn on the activation of transcription factors in BEAS cells. Western blot analyses using phosphospecific antibodies showed that exposure to 500 µM As, Cr, Cu, V, or Zn induced phosphorylation of the transcription factors ATF-2 and c-Jun in BEAS cells (Fig. 4). In keeping with the pattern established by the Western blots and activity assays, As, V, and Zn were the most effective in activating ATF-2 and c-Jun, with smaller increases observed in BEAS cells treated with Cr and Cu (Fig. 4). Phosphorylation of the transcription factor ElK-1, a substrate of ERK, was not detectable in control or metal-stimulated BEAS cell protein extracts (data not shown).
|
To obtain a functional correlate for the acute metal-induced activation of MAPK and transcription factors, the effect of a transient (20-min) exposure to metals on IL-8 expression in BEAS cells was examined. BEAS cells were exposed to 500 µM vehicle, As, Cr, Cu, V, or Zn for 20 min, the cells were then washed, fresh medium was added, and the release of IL-8 into the medium was measured 6 and 24 h after stimulation. As shown in Fig. 5, the same exposure to As, V, or Zn that resulted in MAPK and transcription factor activation induced enhanced IL-8 expression in BEAS cells. This was evidenced by significant increases in IL-8 protein release detected at 6 h, which became significantly more pronounced 24 h after exposure to As, V, or Zn (Fig. 5). Although statistically significant, the effect of As on IL-8 protein synthesis was smaller (2.9 ± 0.87- and 2.3 ± 0.50-fold increase at 6 and 24 h, respectively) compared with that induced by V (7.4 ± 5.3- and 5.4 ± 3.6-fold) and Zn (10.2 ± 4.1- and 13.6 ± 7.2-fold).
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DISCUSSION |
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These studies demonstrate that acute exposure to metals commonly found as ambient air contaminants can induce a rapid activation of three distinct MAPKs, result in the phosphorylation of MAPK-dependent transcription factors, and induce IL-8 expression in the human bronchial epithelial cell line BEAS. Specifically, our findings reveal a consistent pattern in which treatment of BEAS cells with V, As, or Zn was most effective in inducing pronounced MAPK activation, phosphorylation of ATF-2 and c-Jun, and elevated IL-8 release, whereas exposure to Cr and Cu produced generally weaker effects.
Because MAPK activity requires dual phosphorylation, the activation of ERK, JNK, and P38 by certain metals is evidence of phosphorylation of both Tyr and Ser/Thr residues in BEAS cells and thus implicates activation of the dual-specificity MAPK kinases located upstream of the MAPKs in the cascade (53, 55). Therefore, phosphorylation of ERK, JNK, and P38 may be secondary to the activation of their respective MAPK kinases, which, in turn, could imply activation of the next level of kinases, the MAPK kinase kinases. Moreover, the activation of the three MAPKs with consistent relative potency by each metal ion might suggest that the phosphorylation of ERK, JNK, and P38 was induced at a common point in the MAPK cascade. However, no single kinase that can phosphorylate ERK, JNK, and P38 has been described.
A second possible mechanism that may be responsible for metal-induced activation of MAPK in BEAS cells is inhibition of phosphatase activity. Pentavalent and tetravalent V ions are potent inhibitors of protein Tyr phosphatase activity (18) and have previously been shown to activate MAPKs in a variety of cell types (21, 41, 68). Moreover, we have previously shown that treatment of BEAS cells with a V-containing metallic mixture rapidly induces a persistent accumulation of protein Tyr phosphates through a mechanism that involves protein Tyr phosphatase inhibition (50). Similarly, trivalent As reportedly activates JNK and P38 in HeLa cells by inhibiting a dual-specificity Thr/Tyr phosphatase (5), and Zn has been shown to inhibit the receptor Tyr phosphatase HPTP beta (62). Thus the fact that the metals As, V, and Zn, which we report as the most potent activators of MAPKs, are known phosphatase inhibitors suggests that disruption of Tyr phosphate and, possibly, Ser/Thr phosphate homeostasis is a pivotal initiating event in metal-induced activation of MAPKs. Under such a scenario, basal levels of upstream kinase activity, when unopposed by phosphatase activity, would be sufficient to produce an accumulation of MAPK phosphorylation and thereby effect their activation. Additional work will be required to identify specific Tyr, Ser/Thr, or dual-specificity phosphatases in which inactivation increases MAPK activity in bronchial epithelial cells exposed to metals.
The metals Cu and Cr are not known to be phosphatase inhibitors. However, Cu is a transition metal capable of supporting redox cycling and generating reactive oxygen species such as H2O2 (22), which is a potent Tyr phosphatase inhibitor (54) and activator of MAPK (14). Similarly, hexavalent Cr is a potent oxidant and has been shown to activate ERK activity in hepatoma cells through a redox-sensitive mechanism (26) that may involve inactivation of phosphatases. The fact that Cr III and Cr VI appeared to be equipotent activators of MAPKs in BEAS cells may reflect intracellular conversion of the two species (19). Our finding that Fe, the prototypical Fenton metal, did not activate MAPKs in the BEAS cells argues against the involvement of metal-generated reactive oxygen species in activating MAPKs. However, like most cell types, BEAS cells use specialized proteins to bind, transport, and sequester catalytically active free Fe (17). It is possible to speculate that these mechanisms (i.e., lactoferrin, ferritin, transferrin) serve to sequester Fe and prevent it from generating reactive oxygen species that could activate MAPKs. Our finding that exposure to Ni did not produce a significant activation of MAPKs in BEAS cells is in agreement with previous findings that showed that 1 mM Ni was an ineffective stimulus of MAPK activity in 3T3 fibroblasts (20).
To our knowledge, the effect of Cu on MAPKs has not been reported
before. However, we recently determined that Cu can induce cytokine
expression in cultured human bronchial epithelial cells through a
mechanism that involves activation of nuclear factor (NF)-B
(M. W. Frampton, A. J. Ghio, J. M. Samet, J. L. Carson, J. D. Carter, and R. B. Devlin, unpublished observation), suggesting that Cu
may activate signaling pathways such as MAPKs, which may lead to
NF-
B activation (32, 52).
Overall, the findings from the MAPK activity assays correlated well with the Western blot and in-gel kinase results and provided confirmation of a metal-specific activation of ERK2, JNK, and P38 MAPKs in BEAS cells. Interestingly, despite inducing marked phosphorylation of JNK and its substrate c-Jun as well as potently inducing IL-8 expression, Zn exposure caused a relatively modest increase in JNK activity in the BEAS cells (Fig. 3B). The mechanistic basis of this disparity is unknown; however, such a dissociation between JNK phosphorylation and JNK activity suggests a role for an inhibitory process (possibly mediated by hyperphosphorylation), a temporal difference in the kinetics of these events, or the absence of a Zn-specific necessary cofactor in the in vitro assay of JNK activity in immunoprecipitates.
Although the BEAS 2B cell line used in this study was derived by transforming human bronchial cells with an SV40 construct, previous studies have shown excellent functional and mechanistic correlation between BEAS cells and primary cultures of human airway epithelial cells (9, 34, 40, 48, 49). Nonetheless, it is possible that BEAS cell responses to metal exposure differ from those of human airway epithelial cells, and studies aimed at determining the extent to which effects observed in vitro are representative of cellular responses in vivo are needed.
The activation of the distinct MAPKs ERK, JNK, and P38 in human
bronchial epithelial cells exposed to metals may result in cellular
responses such as growth, proliferation, apoptosis, and modulated
inflammatory protein expression (30, 33). The expression of the
cytokines IL-6, IL-8, and TNF- has been shown to be regulated through signaling pathways that involve MAPKs (2, 25, 51) and
activation of the transcription factors ATF-2 and c-Jun (1, 6, 10, 37,
58, 59). Most strikingly, in the present study, the same brief exposure
to As, V, or Zn that induces MAPK activation and transcription factor
phosphorylation was sufficient to effect a subsequent increase in IL-8
expression in BEAS cells. This finding may have profound implications
concerning the effectiveness of clearance mechanisms in protecting
against metal-induced lung injury. Specifically, the finding that
short-term exposure to metals, as might occur with metals leaching from
a metal-laden particle deposited in the airway, can trigger subsequent
IL-8 expression suggests that particle clearance may not occur
sufficiently rapidly to prevent an inflammatory focus from developing.
Although no mechanistic correlation between them was established in this study, the temporal association between MAPK activation, transcription factor phosphorylation, and subsequent cytokine expression provides a strong circumstantial link between these events and suggests a mechanism for enhanced expression of proteins capable of mediating inflammatory pulmonary responses to inhaled metallic compounds in the airway.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the technical assistance of Yaqin He and Susan Burkett.
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FOOTNOTES |
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This work was supported by Environmental Protection Agency Cooperative Agreement 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 trade names constitute endorsement or recommendation for use.
Address for reprint requests: J. M. Samet, Human Studies Division, NHEERL, MD 58D, Research Triangle Park, NC 27711.
Received 3 November 1997; accepted in final form 11 May 1998.
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