Role of extracellular signal-regulated protein kinases in
apoptosis by asbestos and
H2O2
Luis Albert
Jiménez1,
Christine
Zanella1,
Hua
Fung1,
Yvonne M. W.
Janssen1,
Pam
Vacek2,
Colette
Charland3,
Jonathan
Goldberg1, and
Brooke T.
Mossman1
Departments of 1 Pathology,
2 Biostatistics, and
3 Medicine, College of
Medicine, University of Vermont, Burlington, Vermont 05405
 |
ABSTRACT |
Stimulation of cell signaling cascades by
oxidants may be important in the pathogenesis of pulmonary and pleural
diseases. Here, we demonstrate in rat pleural mesothelial cells that
apoptotic concentrations of crocidolite asbestos and
H2O2
induce phosphorylation and activation of extracellular signal-regulated
protein kinases (ERK). Activation of
c-jun-NH2-terminal
protein kinases (JNK)/stress-activated protein kinases was also
observed in response to
H2O2.
In contrast, asbestos caused more protracted activation of ERK without
JNK activation. Both
H2O2-
and asbestos-induced activation of ERK was abolished by catalase.
Moreover, chelation of surface iron from crocidolite fibers or addition
of
N-acetyl-L-cysteine
prevented ERK activation and apoptosis by crocidolite, indicating an
oxidative mechanism of cell signaling. The MEK1 inhibitor PD-98059
abrogated asbestos-induced apoptosis, confirming a causal relationship
between ERK activation and apoptosis. These results suggest that
distinct cell-signaling cascades may be important in phenotypic
responses elicited by oxidant stresses.
protein phosphorylation; oxidants; cell signaling
 |
INTRODUCTION |
EXCESSIVE PRODUCTION of oxidants appears to play a role
in the pathogenesis of a number of pulmonary and pleural disorders, including fibrosing alveolitis, bronchopulmonary dysplasia, emphysema, asthma, acute respiratory distress syndrome, and asbestos-induced diseases (reviewed in Ref. 4). Accordingly, the mechanisms of
oxidant-mediated phenotypic changes in cells of the lung and pleura are
not clearly understood. Moreover, the cell-signaling events eliciting
morphological and functional alterations in target cells are
uncharacterized.
Features of many oxidant-associated diseases include inflammation and
unregulated proliferation of cells, and a dynamic balance between cell
death, mitogenesis, and transformation by oxidative stresses may occur
in lung (2). Apoptosis, a unique type of programmed cell death, may be
important in both resolution and/or promotion of carcinogenesis
and other proliferative diseases (19), but signaling pathways
regulating apoptotic changes in normal cells of the respiratory tract
and pleura are undefined. Our work here focuses on the activation of
extracellular signal-regulated kinases (ERK) and
c-jun-NH2-terminal
protein kinases/stress-activated protein kinases (JNK/SAPK), members of
the family of mitogen-activated protein kinases (MAPK), and their
relationship to apoptosis induced by
H2O2
and crocidolite asbestos, an iron-containing fiber causing inflammation, fibrosis, and cancer (20).
MAPK including the ERK, JNK/SAPK, and p38 are activated in response to
a number of extracellular stimuli (1, 22). Moreover, stimulation of
various MAPK appears to be an upstream event that then leads to
phosphorylation and activation of a number of different target
proteins, including c-Fos and c-Jun, which can dimerize to form
transcription factors, i.e., activator protein-1 (AP-1) (16). Previous
work from our laboratory has demonstrated induction of
c-fos and
c-jun protooncogenes and increased
AP-1 DNA binding in rodent mesothelial cells exposed to asbestos and
other carcinogenic fibers (13, 14). Both
H2O2
and crocidolite asbestos cause transcriptional activation of
c-jun in tracheal epithelial cells, and functional consequences of overexpression of
c-jun are increased cell proliferation
and morphological transformation of this cell type (25). In rat pleural
mesothelial (RPM) cells, asbestos (
5.0
µg/cm2) and
H2O2
(
200 µM) induce apoptosis (5, 11). Moreover, exposure of RPM cells
to asbestos causes phosphorylation of the epidermal growth factor
receptor, preceding activation of ERK (29).
In studies here, we first examined in RPM cells whether asbestos or
H2O2
would activate the ERK or JNK pathways differentially in a dose- and
time-dependent manner. We then used
N-acetyl-L-cysteine (NAC), which increases cellular thiol levels in RPM cells and abrogates
asbestos-induced c-fos and
c-jun expression (15), to determine
whether the redox status of the cell affected crocidolite-induced ERK
activation and apoptosis. We also investigated the effects of exogenous
catalase and chelation of iron from fibers to determine whether ERK
activation by asbestos was due to oxidant generation by fibers. A
synthetic MEK1 inhibitor (8) of the ERK pathway was employed to assess
whether ERK activation was causally related to the development of
crocidolite-induced apoptosis. Studies here are the first to
demonstrate a causal relationship between protracted ERK activation and
apoptosis, a phenomenon related to cell injury and repair, in pleural
cells.
 |
MATERIALS AND METHODS |
Chemicals and asbestos. Reference
samples of National Institute of Environmental Health
Sciences-processed crocidolite asbestos [Na2(Fe3+)2(Fe2+)3Si8O22(OH)2]
were obtained from the Thermal Insulation Manufacturers Association
fiber repository (Littleton, CO). The physicochemical characteristics
of this sample of crocidolite including its fiber dimensions, high iron
content, rodlike structure, and durability have been previously
characterized (14). Desferrioxamine mesylate and Ferrozine
[3-(2-pyridyl)-5,6-bis(4-phenylsulfonic
acid)-1,2,4-triazine] were obtained from Sigma (St.
Louis, MO).
Cell cultures and exposure to test
agents. RPM cells isolated from the parietal pleura of
Fischer 344 rats were grown in Dulbecco's modified Eagle's
medium/Ham's F-12 medium containing 10% fetal bovine serum (both from
GIBCO, Grand Island, NY) and (in µg/ml) 1 hydrocortisone, 25 insulin,
25 transferrin, and 25 sodium selenite (13). Cells were used between
passages 1 and
12. Before addition of test agents,
confluent cultures were switched to 0.5% serum-containing medium for
24 h. Crocidolite asbestos fibers, sterilized at 225°F for
12-15 h, were resuspended in Hanks' balanced salt solution (HBSS)
at 1 mg/ml and triturated 8 times through a 22.5-gauge needle before
their addition to confluent cultures at 5-10
µg/cm2 area of culture dish (13,
14). Confluency of cells in in vitro kinase activity assays is a
prerequisite for low background levels in controls.
H2O2
was diluted in phosphate-buffered saline (PBS) and added to cultures at
a final concentration of 100, 200, or 300 µM. In selected
experiments, RPM cells were preexposed to NAC (10 mM) (Sigma) for 18 h
before the addition of crocidolite (15). In other experiments, catalase
(50 or 500 U/ml; Sigma), heat- or aminotriazole (30 mM)-inactivated
catalase (500 U/ml), and 1% bovine serum albumin (BSA) were added to
cultured cells 1 h before exposure to crocidolite. The phorbol ester
12-O-tetradecanoylphorbol 13-acetate
(Consolidated Midland, Brewster, NY), a positive control for ERK2
activation, was added to cultures from a stock solution of 1 mg/ml in
acetone at a final concentration of 100 ng/ml medium (29). Anisomycin
(Sigma), a positive control for JNK1, was diluted in 0.1 N HCl and
added at a final concentration of 0.6 mM. The synthetic MEK1 inhibitor
PD-98059 was obtained from New England Biolabs (Beverly, MA) (8) and
did not inhibit JNK1 activity induced by
H2O2
in RPM cells (unpublished data).
Chelation of iron on asbestos fibers.
The iron chelators desferrioxamine mesylate and Ferrozine (2 mM) were
dissolved in Ca2+- and
Mg2+-free PBS and added to
asbestos fibers for 24 h according to methods described previously (9).
After centrifugation, fibers were resuspended in HBSS, triturated as
described above, and added to confluent cultures at a final
concentration of 5 µg/cm2 area
of dish. Other cell cultures were exposed to asbestos fibers treated
identically but without addition of iron chelators. In other groups, 1 mM desferrioxamine mesylate was added to medium alone and with
untreated or iron-chelated crocidolite (5 µg/cm2 area of dish) (23).
In vitro kinase activity assays.
Assays for ERK2 or JNK1 activity were performed individually using an
immunoprecipitation assay as described previously (12, 29), employing
either the rabbit polyclonal anti-ERK2 (C-12; 0.1 µg/µl) or
anti-JNK1 antibody (Santa Cruz, CA) at a 1:100 dilution. Glutathione
S-transferase-c-Jun (kindly provided
by Dr. Roger Davis, University of Massachusetts) and myelin basic
protein (Sigma) were used as substrates for JNK1 and ERK2 kinase
activity, respectively. Incorporation of
32P into substrate was detected by
autoradiography. Data were quantitated using a phosphorimager (Bio-Rad,
Hercules, CA) or a Microtex scanning densitometer (see Fig. 6).
Techniques for determination of
apoptosis. Fluorescence-activated cell sorting (FACS)
after propidium iodide staining of DNA was performed, as described by
Bérubé et al. (5), on pooled preparations of detached cells
in medium and those attached to dishes after their removal using
trypsinization. After filtration through a 53-µm nylon mesh filter,
single cells (10,000 gated events per group) were analyzed using an
Epics Elite cytometer (Coulter, Miami, FL) with a 1,024-linear-channel
histogram with Elite software to determine the percentage of cells in
apoptosis. Staining with 4',6-diamidino-2-phenylindole (DAPI) was
used as a second method to detect apoptotic cells; these methods were described previously (5). One thousand cells per coverslip (n = 3/group) were evaluated using
epifluorescence and an ultraviolet excitation filter for the apoptotic
fraction exhibiting chromatin condensation or apoptotic bodies using a
blind coding system. With both FACS and DAPI, crocidolite asbestos at
concentrations
5 µg/cm2 dish
is associated with statistically significant increases
(P < 0.05) in numbers of apoptotic
cells at 24 h after exposure (5).
Statistical analysis. Data from
individual experiments were examined by analysis of variance using
Duncan's procedure to correct for multiple comparisons. All
experiments were repeated in duplicate.
 |
RESULTS |
Addition of
H2O2
to RPM cells induced dose-related and significant increases
(P < 0.05) in ERK2 kinase activity
that diminished over a 4-h time period (Fig.
1). Approximately a 30-fold induction above
control levels was noted with 300 µM
H2O2
at early time points, which decreased with time over a 4-h period to a
10-fold induction.

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Fig. 1.
Extracellular signal-regulated protein kinase (ERK2) activation by
H2O2.
At confluency, rat pleural mesothelial (RPM) cells were exposed to
H2O2
(100-300 µM) for 30 min and 1, 2, and 4 h. Lysates were prepared
and analyzed for ERK2 activity according to MATERIALS
AND METHODS. A: ERK2
activity assay. Cont, control; MBP, myelin basic protein.
B: quantitation of ERK2 activity by
phosphorimaging. Bars (left to
right): open, Cont; hatched, 100 µM
H2O2;
filled, 200 µM
H2O2;
crosshatched, 300 µM
H2O2.
Values are means ± SE for n = 2 lanes/group. All experiments were performed in duplicate; cpm,
counts/min. * P < 0.05 vs.
Cont.
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We previously demonstrated phosphorylation of ERK1 and ERK2 by Western
blot analysis in RPM cells at 8 h after initial exposure to 5 µg/cm2 crocidolite asbestos
(29). To establish whether phosphorylation of ERK proteins resulted in
increased kinase activity and to establish the time frame of responses,
we exposed RPM cells to crocidolite asbestos (5 µg/cm2) for 8, 24, 48, and 72 h. ERK2 activation by asbestos was observed at each of the time points
up to 72 h (Fig. 2).

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Fig. 2.
Activation of ERK2 by crocidolite (Croc) in RPM cells. Confluent RPM
cells were exposed to 5 µg/cm2
Croc for 8, 24, 48, and 72 h and assayed for ERK2 activity using an in
vitro immune complex kinase assay as described previously (29).
A: ERK2 activity assay.
B: quantitation of ERK2 activity by
phosphorimaging. Values are means ± SE for
n = 2 lanes/group. Bars: open, Cont;
filled, Croc. * P < 0.05 vs.
Cont.
|
|
We next examined whether
H2O2
and crocidolite asbestos could also stimulate the JNK/SAPK pathway.
H2O2
(300 µM) activated JNK1 as early as 30 min (Fig.
3). Activation of JNK by
H2O2
was still detected after 4 h of exposure, with maximal
induction at 1 h (13-fold increase in activity compared with untreated
control cells). In contrast, crocidolite asbestos (5 µg/cm2) did not cause
statistically significant increases in JNK1 activation in RPM cells at
any of the time points examined (Fig. 4).

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Fig. 3.
Activation of glutathione S-transferase
(GST)-c-Jun-NH2-terminal protein
kinase (JNK1) by
H2O2.
Cells were exposed to 300 µM
H2O2
or 0.6 mM anisomycin (Aniso) for 30 min and 1, 2, and 4 h. Lysates were
prepared and JNK activity was assessed as described in
MATERIALS AND METHODS.
A: JNK activity assay.
B: quantitation of JNK activity by
phosphorimaging. Values are means ± SE for
n = 2 lanes/group. Bars: open, Cont;
filled, Aniso; hatched,
H2O2.
* P < 0.05 vs.
Cont.
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Fig. 4.
Effects of Croc on JNK activation. RPM cells were exposed to Croc (5 µg/cm2), harvested at various
time periods (30 min to 24 h), and assayed for JNK1 activity as
described in MATERIALS AND METHODS.
Because of the size of this experiment, assays for time points of 30 min to 2 h were performed on different dates, which may account for
relative increases in control activities at later time points.
A: JNK activity assay. B:
quantitation by phosphorimaging. Values are means ± SE for
n = 2 lanes/group. Bars: open,
control; hatched, Aniso; filled, Croc.
* P < 0.05 vs. Cont.
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Because the ERK pathway was stimulated by both agents and exclusively
by asbestos, we focused our attention on whether ERK responses to
agents could be blocked using antioxidants. Figure 5 shows inhibition of
H2O2-induced
ERK2 activation by catalase at both 50 and 500 U/ml medium. The
addition of catalase at 500 U/ml also reduced
(P < 0.05) asbestos-induced ERK
activation (Fig. 6,
A and
B). These effects were not observed
after inactivation of catalase by heat or aminotriazole or after
addition of BSA to medium (Fig. 6C).
Iron chelation of crocidolite fibers by pretreatment with
desferrioxamine and Ferrozine also reduced levels of ERK activity
(P < 0.05) compared with those
observed with unchelated crocidolite (Fig.
7). In contrast, addition of
desferrioxamine alone or simultaneous addition of unchelated
crocidolite and desferrioxamine to medium did not block ERK activation.

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Fig. 5.
H2O2-induced
ERK activation is inhibited by catalase (Cat). RPM cells were
pretreated with either 50 or 500 U/ml Cat for 1 h before addition of
H2O2
(200 µM) for 2 h. ERK2 was immunoprecipitated with anti-ERK2 to
assess kinase activity as described in MATERIALS AND
METHODS.
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Fig. 6.
Cat reduces Croc-induced ERK2 activity.
A: ERK2 activity was determined in
untreated RPM cells (Cont), in cells exposed to Cat (50 and 500 U/ml)
or Croc (5 µg/cm2) alone, and
in cells exposed to Cat (50 and 500 U/ml) for 1 h before addition of
Croc (5 µg/cm2). ERK activity
was examined 24 h after exposure to Croc.
B: quantitation of ERK2 activity
performed using a scanning densitometer. Values are means ± SE for
n = 2 lanes/group.
* P < 0.05 vs. Croc.
C: inactivation of Cat or addition of
1% bovine serum albumin (BSA) does not ameliorate increased ERK2
activity by Croc (5 µg/cm2).
ERK2 activity (24 h) was assessed as described in
MATERIALS AND METHODS. H-cat,
heat-inactivated Cat (500 U/ml); ATZ-cat, 30 mM aminotriazole with Cat
(500 U/ml).
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Fig. 7.
Iron chelation of fibers reduces ERK activity by asbestos. RPM cells
were exposed for 24 h to desferrioxamine (1 mM) alone (Des), 5 µg/cm2 of unchelated Croc fibers
(Croc), Croc fibers treated with desferrioxamine (2 mM) and Ferrozine
(2 mM) [chelated Croc (C-Croc)], unchelated Croc (5 µg/cm2) and desferrioxamine (1 mM) in medium (Croc + Des), or iron-chelated Croc (5 µg/cm2) with desferrioxamine
(1 mM) (C-Croc + Des). ERK2 activity was assessed as described in
MATERIALS AND METHODS.
A: ERK2 activity assay.
B: quantitation of ERK2 activity by
phosphorimaging. Values are means ± SE for
n = 2 lanes/group.
* P < 0.05 vs.
Croc.
|
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Because elevation of glutathione levels protects cells from oxidative
stress and inhibits increased c-fos
and c-jun gene expression by asbestos,
we next examined whether increasing intracellular protein thiol levels
by 10 mM NAC, as described previously in RPM cells (15), could inhibit
crocidolite-associated ERK2 activation. In these studies, activation of
ERK2 by crocidolite was inhibited by pretreatment of RPM cells for 18 h
with NAC, which, when added without asbestos, did not alter ERK
activity (Fig. 8).

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Fig. 8.
N-acetyl-L-cysteine
(NAC) blocks ERK2 activation by crocidolite. RPM cells were exposed to
either 1 or 10 mM NAC for 18 h before addition of Croc (5 µg/cm2). ERK2 activity was
determined 24 h after addition of Croc.
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|
We next determined whether NAC could also ameliorate
crocidolite-induced apoptosis, as evaluated by both flow cytometry and a DAPI staining method (5). When examined by FACS, ~8% of RPM cells
were apoptotic after exposure to crocidolite asbestos for 24 h, in
contrast to the small percentage (0.66%) of apoptosis observed in
untreated controls (Fig.
9A).
Prior addition of NAC for 18 h significantly decreased
(P < 0.05) the percentage of apoptosis observed with crocidolite alone. Compared with controls, crocidolite asbestos caused a threefold increase in number of apoptotic
cells by the DAPI technique, which was also significantly reduced
(P < 0.05) after pretreatment with
NAC (Fig. 9B). In asbestos-exposed groups, the smaller proportion of cells (8% of total cells as opposed
to 15% using the more sensitive in situ DAPI technique) reflects the
fact that cells adhering to long fibers are eliminated in the flow
cytometry assay because they do not go through the membrane filter,
which affords single cells for analysis.

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Fig. 9.
NAC decreases Croc-induced apoptosis as determined by flow cytometry
(A) and
4',6-diamidino-2-phenylindole (DAPI) staining
(B) as described in
MATERIALS AND METHODS.
* P < 0.05 vs. control.
** P < 0.05 vs. Croc.
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To determine whether stimulation of the ERK signaling pathway by
asbestos was directly related to the development of apoptosis, we
pretreated RPM cells for 1 h with a synthetic inhibitor of MEK1, the
upstream activator of ERK. Figure
10A
demonstrates that the stimulation of ERK2 by crocidolite is abrogated
by the MEK1 inhibitor PD-98059 over a 24-h time period, demonstrating
the effectiveness of this approach. Figure
10B shows, using the DAPI technique,
that asbestos-induced increases in apoptosis are significantly diminished (P < 0.05) with the
addition of the MEK1 inhibitor.

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Fig. 10.
The MEK1 inhibitor PD-98059 (MEK1 Inh) diminishes Croc-induced ERK2
activation (A) and apoptosis as
evaluated using DAPI staining (B).
Protocols are outlined in MATERIALS AND
METHODS. A: lanes
(left to
right): Cont, 35 µM MEK1 Inh,
0.1% dimethyl sulfoxide (DMSO), Croc (5 µg/cm2), Croc (5 µg/cm2) + 35 µM MEK1 Inh,
Croc (10 µg/cm2), and Croc (10 µg/cm2) + 35 µM MEK1 Inh.
B: RPM cells were exposed to 35 µM
MEK1 Inh before addition of Croc (5 µg/cm2). Values are means ± SE for n = 2 lanes/group.
* P < 0.05 vs. Cont;
** P < 0.05 vs.
Croc.
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 |
DISCUSSION |
Mammalian MAPK signal transduction pathways including ERK, JNK, and p38
are activated in response to various agents such as growth factors,
phorbol esters, ionizing radiation, and oxidants (1, 12, 24).
Activation of members of the MAPK family lead to the transactivation of
transcription factors, such as AP-1 and Elk-1, that are involved in
expression of genes regulating a battery of distinct cellular events
including apoptosis, proliferation, morphological transformation, and
differentiation (16, 28). We show here that
H2O2
and asbestos activate MAPK signaling pathways in normal
(nontransformed) isolates of rat mesothelial cells, although the
patterns of activation differed with each agent.
In our studies,
H2O2,
an oxidant stress causing cell injury and morphological transformation
of epithelial cells, induced phosphorylation and activation of ERK at
concentrations associated with the development of apoptosis in RPM
cells (5). Activation of ERK proteins by
H2O2
was observed as early as 30 min and declined over a 4-h period.
Previous studies have shown that
H2O2
stimulates ERK activity in several cell types including NIH/3T3 cells,
bovine tracheal myocytes, and PC-12 cells, but these studies have not examined the relationship of ERK activation to consequent cell responses (1, 12, 24). In addition, periods of examination have been
brief (i.e., <60 min), and JNK activation has not been examined
comparatively over time.
Crocidolite asbestos induces the phosphorylation and activation of ERK
proteins, but not of JNK1, in RPM cells. The delayed activation of ERK
by crocidolite fibers compared with activation by
H2O2
may reflect the time period necessary for the sedimentation of fibers
onto cells and/or internalization of sufficient numbers of
fibers to elicit the ERK response. The decreased magnitude of
asbestos-induced responses compared with
H2O2-induced
responses may reflect the localized distribution of fibers in dishes in contrast to the soluble agent
H2O2,
which may affect more widespread numbers of cells. Although
· OH and
H2O2
have been implicated as mediators of asbestos-induced responses because
they are oxidants generated by asbestos fibers or cells phagocytizing
asbestos (reviewed in Ref. 20), other oxidant species may be generated
intracellularly; thus the identification of the reactive oxygen
metabolite(s) responsible for cell signaling events remains obscure.
Mobilization of iron and/or iron on the surface of asbestos
fibers appears to be a key component in crocidolite-associated generation of · OH (26), induction of tumor necrosis
factor-
(TNF-
) (23), single-strand breakage of isolated DNA (6), and lipid peroxidation (27). Iron loading of silicates and other particulates enhances the production of oxidants and cytotoxicity, thereby demonstrating the importance of available iron in these events
(10). Other studies suggest that asbestos causes increases in cell
permeability (21) and oxidative damage to DNA (9) by iron-independent
mechanisms. In studies here, chelation of iron from crocidolite fibers
inhibited ERK activity compared with the increased response observed
with unchelated crocidolite fibers. This finding suggests that surface
iron is a contributing factor in ERK activation by crocidolite fibers.
Because inhibition of ERK activity was not detected when crocidolite
fibers and desferrioxamine were added simultaneously to RPM cells in
medium, extracellular iron may not be as important as available iron on
crocidolite fibers, which may be mobilized intracellularly (18). In
studies here, NAC also inhibited the activation of ERK in pleural
mesothelial cells exposed to crocidolite, further substantiating that
redox status is an important determinant of MAPK signaling. The
addition of catalase to RPM cells ameliorated ERK activation by both
H2O2 and crocidolite, also indicating that active oxygen species play a role
in crocidolite-induced signaling pathways.
The ERK and JNK signaling pathways are stimulated in response to a wide
range of mitogens and environmental stresses. The balance between these
two signaling cascades may be important in phenotypic consequences such
as differentiation and apoptosis. In some studies (7, 28), the
sustained activation of the JNK pathway appears to mediate apoptotic
responses, whereas transient activation of JNK may be linked to cell
proliferation. Our work demonstrates that protracted activation of ERK
by crocidolite and
H2O2
is observed at concentrations of agents inducing apoptosis in RPM cells
and a rat lung epithelial cell line (data not shown), indicating that
effects observed here are not unique to mesothelial cells. In contrast
to
H2O2,
apoptotic concentrations of crocidolite asbestos do not activate the
JNK pathway in either cell type, a result consistent with recent
reports showing that the JNK pathway is not linked to the development
of apoptosis by TNF-
(17) or anti-Fas (7). Our results, using the
specific MEK1 inhibitor PD-98059, substantiate that the ERK pathway is
causally associated with the development of apoptosis by asbestos.
Moreover, the time frame of ERK activation by asbestos and
H2O2,
which precedes demonstration of significantly increased apoptosis in
RPM cells by these agents (5), is consistent with this hypothesis. Our
results and others (7, 17) suggest that different apoptotic stimuli
cause different patterns and kinetics of MAPK signaling, dependent on
cell type. Because apoptosis has recently been indicated as a major
pathway responsible for the resolution of alveolar type II epithelial cells in acute lung injury (3), pharmacological modification of MAPK
pathways may have potential clinical applications in pulmonary and
pleural diseases.
 |
ACKNOWLEDGEMENTS |
We thank Laurie Sabens for technical assistance in the preparation
of the manuscript.
 |
FOOTNOTES |
This work was supported by National Institute of Environmental Health
Sciences Grants R01-ES-06499 and R01-ES-07038 and National Heart, Lung,
and Blood Institute Grant R01-HL-39469.
Address for reprint requests: B. T. Mossman, Dept. of Pathology, Univ.
of Vermont, Burlington, VT 05405.
Received 31 March 1997; accepted in final form 16 July 1997.
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