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INTRODUCTION |
Glass fibers have been popular substitutes for asbestos in the
building industry. The health effects of glass fibers remain to be
fully investigated. Recently, a classifier has been developed to
separate fibers by length using dielectrophoresis that involves the
movement of neutral particles in a gradient electric field (1, 2). The
development of this classifier makes it possible to study the role of
fiber length in toxicity. To this end, we have studied the biological
activity of JM-100 glass fibers on macrophages and found that fiber
length is a very important factor in toxicity and stimulation of
macrophages (3-5). Biological effects of glass fibers of two different
lengths (7 and 17 µm) have been previously studied by our laboratory
(5). Long fibers were more potent than short fibers in the activation
of NF-
B and the induction of
TNF-
1 production.
Macrophages serve as sensors to external stimulation in the defense
system of the body. Reaction of macrophages to glass fibers may be the
first response in the lung when glass fibers are inhaled. The activated
macrophages pass signals to other cells by releasing intercellular
signal mediators, such as cytokines and free radicals (6, 7). In our
previous study, we observed that macrophage engulfed glass fibers and
released TNF-
upon exposure to glass fibers (5). TNF-
is one of
the pro-inflammatory cytokines secreted by macrophages. TNF-
plays
an important role in the pathogenesis of pulmonary fibrosis by
stimulating proliferation of fibroblasts and production of collagen
matrix (6-9).
TNF-
expression is mainly regulated at the transcriptional level.
Transcription of TNF-
is controlled by multiple enhancer elements
such as the
3 site (a NF-
B binding site), cAMP response element
(CRE), and AP-1-binding site. Activities of these enhancers are
regulated by several transcription factors, including NF-
B (5, 10),
AP-1 (11), activated T cell factor (ATF)-2 (12, 13), c-Jun (12, 14),
CRE-binding protein 1 (CREB1) (14), and nuclear factor of activated T
cells (12, 15). It has been widely accepted that both the
3
site and CRE are required for a maximal induction of TNF-
transcription, and a synergy between these two elements is necessary
(12, 14). We have reported previously that the
3 site was involved
in induction of TNF-
by glass fibers (5).
The mitogen-activated protein (MAP) kinases are activated by many
stress signals (16, 17). MAP kinases are serine/threonine protein
kinases that participate in signal transduction of many extracellular
stimuli, including UV light, bacterial derivatives, and growth factors.
The major members in the MAP kinase family are extracellular
signal-regulated kinase (ERK), p38 kinase, and Jun N-terminal kinase
(JNK) (18-20). Activation of these kinases is marked by
phosphorylation of serine/threonine amino residues in their protein
molecule. ERK, p38, and JNK are cytoplasmic proteins. They act as
signal transducers at the end of kinase cascades that mediate signals
from the cell membrane to the nucleus. Activation of ERK, p38, and JNK
leads to induction of transcription factors that in turn regulate
target gene expression in the nucleus. The nuclear proteins, such as
c-Jun, ATF-2, and ElK1, are the major transcription factors that are
regulated by the three MAP kinases. Reactive oxygen species (ROS) have
been shown to act as MAP kinases activators (16, 17, 21).
Our previous study has demonstrated that fiber-induced ROS were
required for TNF-
production in a murine macrophage cell line (5).
The present study focuses on the involvement of MAP kinase signal
transduction pathway in fiber-stimulated TNF-
production by rat
alveolar macrophages. MAP kinases can be activated by ROS in variety of
conditions, including UV light (16, 18, 22), silica (23), and asbestos
exposure (21). Thus, it is likely that MAP kinases will be activated by
glass fiber-induced ROS and that they play a role in glass
fiber-induced TNF-
production. This hypothesis is tested in the
current study. Rat alveolar macrophages were used for analysis of MAP
kinase activity after glass fiber exposure. The following questions are
addressed: (a) Do glass fibers induce TNF-
production in
the rat alveolar macrophages? (b) Do MAP kinases play a role
in glass fiber-induced TNF-
expression? (c) In addition
to NF-
B, are other nuclear factors, such as members of the AP-1
family, involved in glass fiber signaling? (d) How do AP-1
family members regulate the TNF-
gene promoter.
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MATERIALS AND METHODS |
Fibers--
Bulk samples of JM-100 glass fibers (Manville code
100 supplied by the manufacturer) were first milled, aerosolized, and
separated into two length categories using dielectrophoresis (1, 2). The dielectrophoretic classifier was operated in a differential mode so
that fibers with narrow length distributions were extracted in an air
suspension at the end of the classifier. These size-selected fiber
samples were collected on polycarbonate (Nuclepore) filters at rates up
to 1 mg/day. Fibers were scraped off the filters for microscopic
analysis and for biological experiments.
Samples of the length-classified fibers were prepared for size and
count analysis by adding weighed portions of the dusts to freshly
filtered water. These samples were then sonicated, diluted, and
filtered through polycarbonate filters. Measurements of length, width,
and fiber count/mass were made using a JEOL JSM-6400 scanning electron
microscope (4). Measurements at each magnification were referenced to a
National Institute of Standards and Technology electron microscopy
standard rule.
In this study, glass fiber samples with lengths (means ± S.D.) of
7 µm (6.5 ± 2.7) and 17 µm (16.7 ± 10.6), respectively, were used to evaluate fiber effects on macrophages. Concentrations of
the glass fibers used in these experiments were determined as fiber
counts/ml. The glass fiber counts/mg were 3.0 × 108
and 2.0 × 107 for 7- and 17-µm fiber samples,
respectively (4). The glass fiber samples were heat-treated at
120 °C for 2 h and stored under sterile conditions at room
temperature. Before each experiment, the glass fibers were suspended
and sonicated in the complete cell culture medium and then added to cells.
The endotoxin content of the glass fiber samples was measured using the
Limulus amebocyte lysate assay (24). Values ranged from 0.7 to 1.69 endotoxin units/mg. These values are orders magnitude lower than
those found with cotton dust (1000-2000 endotoxin units/mg) or
agriculture dusts (46-4000 endotoxin units/mg) where endotoxin is
thought to play a role (25). The maximum dose of glass fiber used in
this study was 75 µg/ml. Therefore, the maximum endotoxin concentration in this study was 0.013 ng/ml. This dose of endotoxin had
no effect on TNF-
production by macrophages because the minimum effective dose of endotoxin is 1 ng/ml in this experiment system (data
not shown). Therefore, the fiber results reported here cannot be
attributed to endotoxin contamination.
Cells and Reagents--
Pathogen free male Harlan Sprague-Dawley
rats purchased from Hilltop Labs (Scottsdale, PA) were anesthetized
with an intraperitoneal injection of sodium pentobarbitol (45 mg/rat)
and then sacrificed by cutting the renal artery. Following tracheal
cannulation, cold sterile phosphate-buffered saline (PBS)
(Ca2+- and Mg2+-free) was used to lavage the
lungs at a volume of 6 ml for first lavage and 8 ml for subsequent
lavages. Approximately 80 ml of bronchoalveolar lavage fluid was
collected per rat in sterile tubes. Bronchoalveolar lavage cells were
washed twice in PBS by alternate centrifugation and resuspension.
Bronchoalveolar lavage cells from different rats were pooled in sterile
HEPES buffer (145 mM NaCl, 5 mM KCl, 10 mM Na-HEPES, 1 mM CaCl2, 5.5 mM glucose, pH 7.4). Cell counts were performed using an
electronic cell counter equipped with a cell sizing attachment (Coulter
model Multisizer II with a 256C channelizer, Coulter Electronics,
Hialeah, FL). The number of alveolar macrophages was determined by
their characteristic cell diameter (26).
A rat alveolar macrophage cell line (NR8383) was purchased from the
American Type Culture Collection (Masassas, VA). The cells were
maintained in complete medium containing Dubecco's modified essential
medium supplemented with 15% fetal calf serum, 2 mM glutamine, and 100 units/ml penicillin-streptomycin. Supershift antibodies against c-Jun (sc-44 X), c-Fos (sc-52 X), ATF-1 (sc-241 X),
ATF-2 (sc-187 X), CREB1 (sc-186 X), and CREB2 (sc-200 X) were purchased
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The MEK inhibitor
(PD 98059; catalog number 513000), which prevents phosphorylation of
ERK by MEK, and p38 inhibitor (SB203580; catalog number 559389) were
purchased from Calbiochem-Novabiochem Corporation (San Diego, CA). PD
98059 and SB 203580 were dissolved in Me2SO and used
to treat cells with a final Me2SO concentration of 0.1%. In all experiments where the inhibitors were used, Me2SO
was used at the same concentration in the controls.
TNF-
ELISA--
Rat alveolar macrophages were plated in a
96-well plate at 1 × 105 cells/well in 200 µl of
sterile Eagle's modified essential medium supplemented with 10%
heat-inactivated fetal bovine serum, 1 mM glutamine, 100 units/ml penicillin/streptomycin, and 10 mM HEPES at pH
7.2. After a 2-h incubation at 37 °C, the adherent cells were washed
three times with warm sterile PBS and then cultured in 200 µl of
fresh Eagle's modified essential medium. Before exposure to glass
fibers, some cells were pretreated with MAP kinase inhibitors for
1 h. The cell culture supernatant was harvested after overnight exposure to glass fibers at a cell to fiber ratio of 1:5, which resulted in an optimal TNF-
production (5) and then combined from
triplicated wells. The supernatant was diluted 20-fold and used in the
TNF-
ELISA according to the manufacturer's instruction. An ELISA
kit (catalog number KRC3010-SB) from BIOSOURCE
International (Camarillo, CA) was used to determine rat TNF-
.
MAP Kinase Phosphorylation Assay--
Rat alveolar macrophages
were plated in a 6-well plate at 3 × 10 6/well in 2 ml of Eagle's modified essential medium. After a 2-h incubation at
37 °C, the adherent cells were washed three times with warm sterile
PBS and then exposed to glass fibers (cell to fiber ratio of 1:5) in 2 ml of fresh Eagle's modified essential medium. After a 2-h exposure,
cells were washed once in PBS and lysed in 200 µM lysis
buffer for Western blot assay. Activation of ERKs, JNKs, and p38 kinase
was determined by their phosphorylation status detected with
phospho-specific MAP kinase antibodies. The ERK MAP kinase assay kit
(catalog number 9800) and the p38 MAP kinase assay kit (catalog number
9820) from New England Biolabs, Inc. (Beverly, MA) were utilized in
this study. Phospho-specific (catalog number sc-6254) and
nonphospho-specific (catalog number sc-1648) anti-JNK antibodies from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) were used to detect JNK
activity. The signal in Western blot was quantitated using a
densitometer and normalized for protein loading.
Gel Shift Assay--
A CRE binding sequence
(
112TCCAGATGAGCTCATGGGTT
92) in the TNF-
promoter was used to synthesize an oligonucleotide for the CRE probe.
The AP-1 binding element in the collagenase gene promoter was used as
an AP-1 probe to examine the AP-1 binding activity (27). The
double-stranded probe was labeled with [32P]ATP (Amersham
Pharmacia Biotech) using the T4 kinase (Life Technologies, Inc.). The
nuclear extracts were prepared by a three-step procedure (27). The
harvested cells were treated with a lysis buffer. The collected nuclei
were washed once in a washing buffer and then treated with an
extraction buffer. After centrifugation at 14,000 rpm for 5 min, the
supernatant was harvested as the nuclear protein extract and stored at
70 °C. The protein concentration was determined using a BCA
protein assay reagent (Pierce). The DNA-protein binding reaction was
conducted in a 24-µl reaction mixture including 1 µg of poly(dI-dC)
(Sigma), 3 µg of nuclear protein extract, 3 µg of bovine serum
albumin, 4 × 104 cpm of 32P-labeled
oligonucleotide probe, and 12 µl of reaction buffer (24% glycerol,
24 mM HEPES, pH 7.9, 8 mM Tris-HCl, pH 7.9, 2 mM EDTA, and 2 mM dithiothreitol) (27). In some
cases, the indicated amount of double-stranded oligomer was added as a
cold competitor. The reaction mixture was incubated on ice for 10 or 20 min (with or without an antibody) in the absence of radiolabeled probe. After addition of the radiolabeled probe, the mixture was incubated for
20 min at room temperature and then resolved on a 5-6% acrylamide gel
that had been prerun at 170 V for 30 min with 0.5 × TBE buffer. The loaded gel was run at 200 V for 90 min, then dried, and placed on
Kodak X-Omat film (Eastman Kodak, Rochester, NY).
Transfection Assay--
The TNF-
reporter gene vector used in
this study was a gift of Dr. Fan at the Scripps Research Institute (La
Jolla, CA) (14). The luciferase vector contains a promoter fragment
(
615/+15) of the human TNF-
gene. The NR8383 cells (1 × 106/well) were plated in 24-well plates for 16 h
before transfection. The TNF reporter DNA (0.5 µg) and a
cytomegalovirus
-galactosidase expression vector (0.1 µg) were
delivered into the cells by LipofactAMINE reagent (catalog number
18324-020; Life Technologies, Inc.). After transfection, the cells were
washed once in PBS solution and cultured in 1 ml of the complete medium
at 37 °C for 24 h. After being exposed to glass fibers for an
additional 16 h, the cells were harvested for the reporter assay.
The luciferase activity was determined using a luciferase assay kit
(Promega, Madison, WI) and then normalized by the internal control
-galactosidase and the protein content.
Data Analysis--
The data for TNF-
production and
activation of the TNF-
promoter were presented as mean values ± standard deviations of three individual experiments. Results were
analyzed by the Student's t test at a confidence level of
p
0.05.
 |
RESULTS |
Inhibition of TNF-
Production by MAP Kinase Inhibitors in Rat
Alveolar Macrophages--
In our previous study, murine peritoneal
macrophages (Raw 264.7 cells) were used to study TNF-
production
after exposure to size-classified glass fiber samples (5). The present
study attempted to elucidate a role of MAP kinases in glass
fiber-induced TNF-
production using rat alveolar macrophages. Short
(7 µm) or long (17 µm) glass fibers were used to stimulate TNF-
production by rat alveolar macrophages, and TNF-
protein was
determined by an ELISA after a 16-h exposure to the glass fibers. The
results show that glass fibers can induce TNF-
production in rat
alveolar macrophages and that short and long fibers exhibited different stimulatory potencies (Fig.
1A). The long fiber sample was
twice as potent in stimulation of TNF-
production compared with the short fiber sample (7 µm length).

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Fig. 1.
TNF- production in
rat alveolar macrophages exposed to size-selected glass fibers.
The cells were plated in 96-well plates at 1 × 105
cells/well in 200 µl of culture medium and then exposed to glass
fibers at a cell to fiber ratio of 1:5 (equal to 75 µg/ml for long
fiber and 8.7 µg/ml for short fiber). TNF- was determined 16 h later in the cell free supernatant by ELISA. Each bar represents mean
value ± S.D. from three independent experiments. A,
induction of TNF- by long verses short glass fibers. * indicates a
significant (p < 0.001) increase in production of
TNF- induced by short fibers compared with the untreated cells. + indicates a significant increase in TNF- production induced by long
fibers compared with short fibers. B, inhibition of TNF-
production by MAP kinase inhibitors. Cells were pretreated with a p38
inhibitor (SB203580, 2 µM) or an MEK inhibitor (PD98059,
40 µM) for 1 h, then exposed to glass fibers. *
indicates a significant (p < 0.001) decrease in
fiber-induced production of TNF- by in the presence of a p38 or an
MEK inhibitor.
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It is known that MAP kinases are involved in cell stress response (16).
Because ROS were involved in glass fiber-induced TNF-
production
(5), it is possible that the glass fibers activate MAP kinases in
macrophages. The role of MAP kinases was explored by using specific MAP
kinase inhibitors. The cells were pretreated with a p38 inhibitor
(SB203580) or a MEK inhibitor (PD98059) for 1 h before exposure to
glass fibers. SB203580 and PD98059 were added to the cell culture at a
final Me2SO concentration of 0.1%. Me2SO was
used as a control. The results show that TNF-
production was
decreased by both inhibitors. The p38 or MEK inhibitor resulted in 60 or 40% inhibition, respectively (Fig. 1B). This inhibition
was not from Me2SO because Me2SO did not
exhibit any inhibitory effect at this concentration. No cell toxicity
was observed by LDH assay. Similar results were found with long fibers as well (data not shown). These results suggest that MAP kinases might
be required for TNF-
production by macrophages in response to
glass fibers.
Activation of MAP Kinases by Glass Fibers--
Three MAP kinases,
p38, ERK, and JNK, were analyzed in this study. Activation of these
three kinases is associated with phosphorylation status of serine or
threonine residues. Phospho-specific kinase antibodies were employed to
detect phosphorylated p38, ERK, or JNK using Western blot analysis. The
rat alveolar macrophages were exposed to glass fiber for 2 h, and
the whole cell lysate was used for the phosphorylation assay. The
protein-containing membrane was blotted with phospho-specific p38
antibody, which can specifically recognize the dual phosphorylated
threonine 180 and tyrosine 182 in the p38 MAP kinase protein. As shown
in Fig. 2A, exposure of rat
alveolar macrophages to glass fibers resulted in phosphorylation of p38
MAP kinase. After being stripped, the membrane was reblotted with a
regular a p38 antibody, which revealed the total p38. The
phosphorylation signal was normalized for total p38 protein. The result
shows that glass fibers activated p38 MAP kinase and that long fibers
expressed a stronger activity than short fibers. Phosphorylation of p38
was induced 7-fold by long and 3.5-fold by short glass fibers compared
with the untreated group (Fig. 2B).

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Fig. 2.
Glass fiber-induced activation of MAP
kinases. Phosphorylation status of MAP kinases, p38, ERK, and JNK,
was analyzed by Western blot. The rat alveolar macrophages were plated
in 6-well plates at 3 × 106 cells/well and then
treated with glass fibers at a cell to fiber ratio of 1:5 for 2 h.
The whole cell lysate was used in the experiment. Phosphorylation
signals and protein abundance signals of p38, ERK and JNK are presented
in A, C, and E, respectively. The
normalized densitometric results are plotted in B,
D, and F, respectively.
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ERK and JNK MAP kinases were analyzed in a similar manner. The results
indicate that ERK was also activated by glass fibers (Fig.
2C). Phosphorylation of ERK was induced 14-fold by long and
7-fold by short glass fibers (Fig. 2D). In contrast, JNK
activity was not activated by glass fibers (Fig. 2, E and
F). These results suggest that p38 and ERK may be major
components in the signal transduction pathway for TNF-
induction by
glass fibers.
Association of MAP Kinase with Activation of the TNF-
Gene
Promoter--
The above results indicate that p38 and ERK may be
involved in induction of TNF-
production by glass fibers. Because
TNF-
expression is controlled at the transcriptional level, the role of p38 or ERK in activation of the gene promoter by glass fibers was
examined. The promoter activity of the TNF-
gene was studied by
transient transfection of rat alveolar macrophage cells (NR8383). A
luciferase reporter vector that is controlled by a wild type TNF-
gene promoter was used for this purpose. Macrophages were transfected
by the plasmid vector and then exposed to glass fiber samples. The
reporter assay indicates that the TNF-
gene promoter was activated
by both short and long glass fibers (Fig.
3A). However, long fibers were
nearly three times as potent as short fibers. Long fibers induced a
20-fold increase, whereas short fibers induced a 7-fold increase in the
promoter activity.

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Fig. 3.
Activation or inhibition of the
TNF- gene promoter in rat alveolar
macrophages. The transient transfection assay was used to study
the gene promoter activity of TNF- in NR8383 cells. The cells were
transfected with a luciferase reporter (0.5 µg/sample) controlled by
a TNF- gene promoter and then treated with the fibers for 16 h
at a cell/fiber ratio of 1:5. The reporter activity in the cell lysate
was determined using a luminometer, and the reading was normalized for
the amount of protein and an internal control of -galactosidase.
Each bar represents the mean value ± S.D. of the
reporter activities from three independent assays. A,
induction of the promoter activity by glass fibers. * indicates a
significant (p < 0.001) increase in the promoter
activity stimulated by short fibers (7 µm) compared with that of the
untreated cells. + indicates a significant (p < 0.001)
increase in the promoter activity induced by long fibers (17 µm)
compared with short fibers. B, inhibition of the promoter
activity by MAP kinase inhibitors. P38 (SB203580) and MEK (PD98059)
inhibitors were used at 2 and 40 µM to inhibit the long
fiber (17 µm) induced promoter activity. * indicates a significant
(p < 0.001) decrease in the promoter activity induced
by long fibers (17 µm) in the presence verses absence of
inhibitor.
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The role of p38 or ERK in activation of the TNF-
promoter was
examined by using specific p38 or MEK inhibitors. SB203580 and PD98059
were added to the cell culture at a final Me2SO
concentration of 0.1%. Me2SO was used as a control.
Pretreatment of the transfected cells with a p38 inhibitor (SB203580)
or an MEK inhibitor (PD98059) significantly reduced the TNF-
gene
promoter response to long glass fibers (Fig. 3B). Similar
results were found with short fibers as well (data not shown). Each of
the inhibitors resulted in approximately a 70% loss of the promoter
activity induced by long fibers. These data provide evidence that p38
and ERK MAP kinases may mediate transcriptional activation of the
TNF-
gene in alveolar macrophages in response to glass fibers.
Induction of AP-1 and CRE DNA-Protein Complexes--
p38 and ERK
MAP kinases regulate target gene transcription through activation of
nuclear proteins such as c-Fos, c-Jun, and ATF-2 (18). Nuclear proteins
c-Fos and c-Jun are two subunits of the transcription factor AP-1. In
addition to interaction with the AP-1 site, c-Jun is able to activate a
CRE element by forming homodimer or heterodimer with ATF-2. Because
both AP-1 and CRE elements are enhancers in the TNF-
gene promoter,
it is quite possible that p38 and ERK MAP kinases induced the TNF-
gene promoter through AP-1 and CRE elements. The protein binding
activities of AP-1 and CRE elements were investigated in nuclear
extracts of alveolar macrophages using an EMSA. The results show
that DNA binding activity of AP-1 was significantly induced by glass
fibers and that long fibers exhibited a stronger activity than short fibers (Fig. 4A, lanes
1-3). The binding specificity and protein nature of the AP-1
complex were confirmed by oligonucleotide competition and antibody
supershift. The AP-1 complex was specifically removed by cold AP-1
probe (Fig. 4A, lane 5) or anti-Jun antibody
(Fig. 4B, lane 2) but was not changed by a
NF-
B probe (Fig. 4A, lane 6) or an ATF-2
antibody (Fig. 4B, lane 3).

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Fig. 4.
Activation of the DNA binding activity of
AP-1 and CRE by glass fibers. The AP-1 activity was examined by
EMSA after the cells were exposed to the fibers for 4 h as stated
under "Materials and Methods." The cell to fiber ratio was 1:5.
A, induced AP-1 activity. Lane 1 contained the
control nuclear protein; lanes 2 and 3 contained
nuclear protein from the short and long fiber-treated cells,
respectively. Competition assay was carried out with the nuclear
protein from long fiber-treated cells (lanes 4-6) and 100 ng of cold probe. B, protein nature of the complex
determined by antibody supershift. The nuclear protein of long
fiber-treated cells was used. Antibodies to c-Jun (lane 2)
and ATF-2 (lane 3) were used. C, induced CRE
binding activity. There were three major complexes formed by incubation
of the CRE probe with the nuclear protein. The short fibers (lane
2) and long fibers (lane 3) induced complex A. Binding
specificity of the complexes was analyzed with oligonucleotide
competition. The unlabeled CRE probe (100 µg) was used in lane
4 as a specific competitor. The same amount of unlabeled 3
probe was used in lane 5 as a nonspecific competitor.
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The CRE probe formed three DNA-protein complexes (A, B and C) in the
macrophage nuclear extracts as revealed by the EMSA (Fig. 4C
and Fig. 5). Treatment with glass fibers
resulted in an increase in complex A and a marginal increase in complex
B (Fig. 4C, lanes 2 and 3). A change
in complex C was not significant. Specificity of the three complexes
was demonstrated by oligonucleotide competition. Unlabeled CRE probe
effectively competed with the radiolabeled probe in the three
complexes, whereas unlabeled k3 probe had no competitive effect on
complex A and B (Fig. 4C, lane 4 and
5). However, the unlabeled k3 probe competed with the CRE
probe in complex C. Other experimental results suggest that the k3
probe is able to bind specifically to proteins in complex C (data not shown).

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Fig. 5.
Characterization of c-Jun binding to
CRE. The protein nature of CRE complexes was analyzed by antibody
supershift in an EMSA. Antibodies used are listed at the top
of each lane. A, c-Jun and CREB1 binding to CRE.
The nuclear protein from the control cells was used. The nuclear
proteins c-Jun and CREB1 were two major CREB binding proteins, because
the corresponding antibodies decreased these complexes. B,
increased c-Jun binding after exposure to glass fibers. The nuclear
protein from long fiber-treated cells was used. c-Jun became the major
binding protein in CRE after glass fiber treatment, because only c-Jun
antibody removed complexes A and B.
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Binding of c-Jun to CRE--
It has been reported that c-Jun and
ATF-2 were responsible for activation of the CRE element in macrophages
and for induction of TNF-
transcription (14), suggesting that c-Jun
and ATF-2 might be involved in formation of the CRE complexes. To
examine the possibility, five antibodies to CRE binding proteins were used in supershift assays. They were CREB1, CREB2, c-Jun, c-Fos, and
ATF-2 antibodies as indicated in Fig. 5. Nuclear proteins from
untreated cells (Fig. 5A) and fiber-exposed cells (Fig.
5B) were analyzed in the supershift assay. The results
revealed that glass fibers were able to change the components of the
CRE complexes. In the control cells, complex A is formed by c-Jun
because only c-Jun antibody was able to remove it completely (Fig.
5A, lane 4). Both c-Jun and CREB1 proteins are
involved in formation of complex B because antibodies to either c-Jun
or CREB1 partially reduced this complex (Fig. 5A,
lanes 2 and 4). CREB1 is involved in complex C
because CREB1 antibody partially reduced the complex (Fig.
5A, lane 2).
In the glass fiber-treated cells, DNA binding activity was increased in
both complex A and complex B (Fig. 4C). It may be noted that
c-Jun antibody completely removed complex A and dramatically decreased
complex B. This is in contrast to the fact that c-Jun antibody only
weakly reduced complex B in the control cells (Fig. 5A).
This change indicates that more c-Jun protein binds to CRE element
after glass fiber exposure. In the unstimulated cells, CREB1 was
involved in formation of complexes B and C. Interestingly, in the glass
fiber-stimulated cells, no CREB1 protein was detected in complexes B
and C because CREB1 antibody did not reduce either of the two complexes
(Fig. 5B, lane 2). These results were consistent in the repeated experiments. The supershift results suggest that although the binding pattern of CRE was similar before and after fiber
exposure, proteins in the complexes B and C have been changed by the
fiber stimulation. c-Jun protein gained more binding activity after
cell activation. Complex C was also reduced by c-Jun antibody, indicating that c-Jun was involved in complex C after fiber
stimulation. Complex C was not reduced by CREB1 antibody, suggesting
that CREB1 was not in the complex after fiber stimulation. These
results imply that in the activated cells, c-Jun substituted for CREB1 in formation of complex C. The supershift results do not support the
involvement of CREB2, c-Fos, or ATF-2 in the formation of CRE complexes.
Enhancement of TNF-
Promoter Activity by c-Jun--
The above
results suggest that glass fibers induce TNF-
transcription through
a CRE or AP-1 element and that nuclear protein c-Jun might play a major
role in this activation. To test this hypothesis, functional analysis
of the TNF-
promoter was carried out by mutation and cotransfection.
In the mutation analysis, the CRE element was inactivated by base pair
substitution in the DNA sequence of the TNF-
promoter (14). The
promoter activity was then examined in rat alveolar macrophages
(NR8383) in a transient transfection assay. The results show that
mutation of CRE resulted in a significant loss of the promoter response
to the glass fibers (Fig. 6A).
Compared with the wild type promoter, the mutated promoter lost ~87%
of its inducibility to both short and long fibers. This suggests that
CRE may be required for fiber-induced TNF-
transcription.

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Fig. 6.
Requirement of the CRE element and c-Jun
binding in the promoter. Transient transfection was carried out in
NR8383 cells. Each bar represents the means ± S.D. of the
normalized luciferase activity of three independent experiments.
A, effect of CRE mutation on the TNF- gene promoter. A
mutated promoter, in which the CRE element was inactivated, was
compared with the wild type (WT) promoter in response to
glass fibers. * indicates a significant (p < 0.001)
suppression of the promoter activity compared with the wild type.
B, effect of c-Jun cotransfection on the TNF- gene
promoter. A c-Jun expression vector was cotransfected with the reporter
at a DNA weight ratio of 1:2. * indicates a significant
(p < 0.001) increase in the TNF- gene promoter
activity in the presence of c-Jun expression vector. indicates a
significant suppression of the mutated promoter in response to c-Jun
compared with the wild type.
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An increase of c-Jun expression by a c-Jun expression vector resulted
in activation of the TNF-
promoter. Because CRE is required for
glass fiber-induced TNF-
promoter activity, and c-Jun is a main
protein bound to the element, it is quite possible that c-Jun is
involved the activation of the CRE element. To verify this hypothesis,
c-Jun protein level was increased in the assay system by cotransfection
of a c-Jun expression vector into NR8383 cells. The TNF reporter
activity was investigated under this condition. The result shows that
c-Jun increased the transcriptional activity about 4-fold in the wild
type promoter but only 1-fold in the CRE mutated promoter (Fig.
6B). This indicates that CRE element is required for c-Jun
activity on the gene promoter. The residual promoter activity after CRE
mutation might be contributed by an AP-1 binding site in the TNF-
gene promoter, which remained functional in the CRE-mutated promoter.
Inhibition of c-Jun Activity by p38 and ERK Inhibitors--
MAP
kinase inhibitors SB203580 and PD98059 decreased TNF-
production in
response to glass fibers (Fig. 1B). The promoter response
represents the end result of inhibitor effects on sequential reactions
of the signal transduction pathway controlling TNF-
production.
Because protein binding activities of the AP-1 and CRE elements are
controlled by MAP kinases, p38 and MEK inhibitors should decrease
protein binding activities of the two elements, leading to inhibition
of the TNF-
promoter. To this end, effects of MAP kinase inhibitors
were examined on phosphorylation of ERK, protein binding activities of
AP-1 and CRE elements, and activity of the TNF-
promoter. The
phosphorylation status of ERK was determined by Western blot (Fig.
7A). The results demonstrate
that PD98059 specifically inhibited ERK phosphorylation (Fig. 7,
A and B). Consistent with these results, protein
binding activities of the AP-1 and CRE elements were decreased in the
nuclear extracts from cells treated with SB203580 or PD98059 (Fig. 7,
C and D). These results are in line with data
that the wild type TNF-
gene promoter was inhibited by the two MAP
kinase inhibitors (Fig. 3B). Together, these results
strongly support that glass fibers activate MAP kinase pathways that,
in turn, lead to initiation of TNF-
transcription through induction
of c-Jun DNA binding.

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Fig. 7.
Inhibition of ERK phosphorylation and c-Jun
DNA binding by MAP kinase inhibitors. A, inhibition of
ERK phosphorylation. Phosphorylation of ERK was examined by Western
blot analysis. The rat alveolar macrophages were plated in a 6-well
plate at 3 × 106 cells/well. After pretreatment with
MAP kinase inhibitor PD98059, the cells were exposed to long glass
fibers at a cell to fiber ratio of 1:5 for 2 h. The whole cell
lysate was used in the experiment. Phosphorylation signals and protein
abundance of ERK are presented. B, the normalized results of
gel in A. In the DNA binding assay, nuclear protein was made
from the inhibitor-treated cells. C, DNA binding activity of
c-Jun with AP-1 probe. D, DNA binding activity of c-Jun with
CRE probe.
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DISCUSSION |
Activation of MAP kinases by glass fibers might be due to
phagocytosis by macrophages. Our previous studies have shown that macrophages could engulf glass fibers (4, 5). This phagocytosis is
influenced by fiber length. Short glass fibers (7 µm) were completely
engulfed, whereas long fibers were only partially engulfed by
macrophages. Macrophages generate ROS upon engulfment of short fibers.
It is likely that macrophage would generate more ROS during frustrated
phagocytosis of long fibers. Although ROS may activate all three MAP
kinases, p38, ERK, and JNK, the pattern of activation is dependent on
the stimulant (16, 17). For example, we have observed that silica
particles only induced activation of p38 and ERK, but not JNK (23). The
present study demonstrates that p38 and ERK are the main MAP kinases
activated by glass fibers.
Our study suggests that c-Jun mediates the downstream signal of p38 and
ERK. Although JNK has been proposed as a c-Jun kinase, we did not
observe significant activation of JNK in rat alveolar macrophages
exposed to glass fibers. In this study, activation of p38 and ERK was
associated with an enhanced c-Jun DNA binding activity at both CRE and
AP-1 sites, and suppression of p38 and ERK led to a decreased DNA
binding of c-Jun. These results suggest that p38 and ERK MAP kinases
may play a role in regulation of c-Jun activity, although it is not
known how p38 or ERK phosphorylate c-Jun. Our observations support the
view that p38 is required for c-Jun activity in the activation of
TNF-
gene promoter (28). One possibility is that p38 induces c-Jun
expression (29, 30).
The CRE element is required for induction of TNF-
transcription by
several stimuli in various types of cells. In T cells, CRE is activated
by the T cell receptor signal induced by phorbol 12-myristate
13-acetate or ionomycine (12, 15). In B cells, CRE is activated by CD40
ligation (13). In macrophages, CRE is activated by lipopolysaccharide
(LPS) (14). In fibroblast cells, CRE can be activated by the cytokine
TNF-
(28). Activity of the CRE element is mediated by different
transcription factors in distinct cell types. In the T, B, and
fibroblast cells, the nuclear factors c-Jun and ATF-2 have been
reported as the major binding proteins of CRE (12, 13, 28). In
macrophages, the binding proteins have been reported to be c-Jun and
CREB1 (14). Consistent with these conclusions, CREB-binding protein is
required for transcriptional induction of TNF-
by T cell receptor
(31). CREB-binding protein, a homolog of p300 (32), is a
transcriptional adaptor that integrates signals from many
sequence-specific activators including c-Jun, ATF-2, and CREB via
direct protein-protein interactions (33). It is known that CREB-binding
protein/p300 is a histone acetyltransferase (34).
The present study provides evidence that CRE was required for TNF-
transcription induced by glass fibers. This evidence includes: (a) Protein binding at the CRE element was associated with
fiber-induced TNF-
production (Figs. 1B and 7).
Nuclear protein c-Jun was the major CRE-binding protein in rat alveolar
macrophages. An increase or decrease in c-Jun binding activity led to
activation or inhibition of TNF-
transcription. When c-Jun binding
was induced by glass fibers (Figs. 4C and 5), TNF-
production and promoter activity were increased (Figs. 1A
and 3A). When c-Jun binding was decreased by MAP kinase
inhibitors (Fig. 7, C and D), glass
fiber-induced TNF-
production and the promoter activity were blocked
(Figs. 1B and 3B). (b) The CRE element
is required for TNF-
promoter activation by glass fibers (Fig. 6). A
mutation that abolished protein binding at the CRE element resulted in
a loss of promoter response to glass fibers, indicating that glass
fibers may regulate TNF-
transcription through the CRE element.
(c) The CRE element is required for the c-Jun effect on the
TNF-
gene promoter. Cotransfection of a c-Jun expression vector led
to a dramatic increase in transcription activity in the wild type
promoter but only weak transcription activity in the CRE
mutated-promoter (Fig. 6B). This suggests that the CRE
element is the major trans-acting site of nuclear factor c-Jun in the
TNF-
gene promoter. The AP-1 binding site may be a weak enhancer in
the promoter. The above evidence strongly supports the hypothesis that
glass fibers activate the TNF-
gene promoter through induction of
DNA binding activity of c-Jun at the CRE element in rat alveolar
macrophages. This is consistent with c-Jun activity in human
monocyte/macrophage U937 and THP-1 cells, in which c-Jun activates the
TNF-
CRE element in response to stimulation by TNF-
or LPS (14,
35).
Characterization of DNA-binding proteins of the CRE element suggests
that, in addition to c-Jun, nuclear factor CREB1 also binds to CRE.
CREB1 was associated with the CRE element in untreated alveolar
macrophages (Fig. 5A). CREB1 binding activity was reduced by
glass fiber exposure, and this change was associated with an increased
binding of c-Jun (Fig. 5B). This indicates that c-Jun is
responsible for activation of the CRE element and that CREB1 may not be
important in the glass fiber signaling pathway or might be a repressor
protein. This is different from CREB1 activity in the LPS signal
pathway, which induces DNA binding of CREB1 (14). Our data suggest that
ATF-2 does not bind to the CRE element in rat alveolar macrophages.
This result is in line with findings reported in human macrophages
(14). Taken together, these results suggest that: (a) the
signaling pathways of glass fibers and LPS are distinct and
(b) the regulatory mechanisms of TNF-
transcription in
macrophages and T cells are different. ATF-2 does not bind to the
TNF-CRE element in macrophages.
The AP-1-binding site (
65/
59) in the TNF-
gene promoter is
another enhancer where c-Jun can regulate transcription (11). The
importance of this AP-1-binding site was verified by mutation studies
on the promoter. Deletion of the AP-1 site significantly reduced both
basal and phorbol 12-myristate 13-acetate-activated TNF-
promoter
activity in U937 cells (11) but only marginally decreased LPS-induced
TNF-
promoter activity (14). In the present study, c-Jun was able to
induce a 1-fold increase in the TNF-
promoter with a mutated CRE
element (Fig. 6B). Because the AP-1 binding site remains
intact in the a CRE-mutated promoter, the 1-fold induction might be a
result of activation of the AP-1 binding site by c-Jun. Glass fibers
induce AP-1 binding activity in rat alveolar macrophages (Fig.
4A), and this is associated with a detectable response of
CRE-mutated promoter to glass fibers (Fig. 6A). These data
indicate that the AP-1 site is involved in the response to c-Jun when
rat macrophages were exposed to glass fibers.
The CRE and AP-1 sites may have a functional synergy in activation of
TNF-
transcription. Although it has been reported that in the
TNF-
promoter, CRE synergizes with the NF-
B binding site (
3
site) (10, 14), it is not clear whether CRE synergizes with the AP-1
site. AP-1 is a heterodimer formed by two subunit proteins, c-Jun and
c-Fos in most cases. Dimerization of the two subunits is required for
stable DNA binding activity. Expression of the c-Fos gene is controlled
by the serum response element. MAP kinases, including p38 kinase, ERK,
and JNK, are able to induce c-Fos expression through serum response
element (18-20). Therefore, inhibition of p38, ERK or JNK would lead
to an inhibition of AP-1 activity. In this study, p38 and ERK
inhibitors both reduced AP-1 DNA binding activity (Fig. 7C).
The inhibition may result from two effects: (a) inhibition
of c-Fos expression that may explain effect of ERK inhibition and
(b) inhibition of phosphorylation of c-Jun protein, which
decreases c-Jun activities such as the DNA binding activity and
trans-activating activity at the AP-1 binding site. If the AP-1 element
synergizes with the CRE element, inhibition of either element would
lead to a dramatic transcriptional inhibition. This may explain the
inhibitory effect of the MAP kinase inhibitors.
In summary, we observed that glass fibers are able to induce TNF-
production in rat alveolar macrophages. In combination with our
previous findings, we conclude that this induction may be dependent on
at least two signaling pathways: NF-
B and MAP kinase. The present
study demonstrated that glass fibers activate p38 and ERK and in turn
activate TNF-
transcription. The nuclear factor, c-Jun, mediates MAP
kinase signals and induces TNF-
transcripion through two promoter
elements, CRE and AP-1. Long fibers are more potent than short fibers
in the activation of MAP kinase, c-Jun DNA binding, and TNF-
transcription.