1 Department of Molecular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871; and 2 National Kinki Chuou Hospital for Chest Disease, Sakai, Osaka 591-8555, Japan
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ABSTRACT |
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To elucidate the pathophysiology of
pulmonary fibrosis, we investigated the involvement of p38
mitogen-activated protein kinase (MAPK), which is one of the major
signal transduction pathways of proinflammatory cytokines, in a murine
model of bleomycin-induced lung fibrosis. p38 MAPK and its substrate,
activating transcription factor (ATF)-2, in bronchoalveolar lavage
fluid cells were phosphorylated by intratracheal exposure of bleomycin,
and the phosphorylation of ATF-2 was inhibited by subcutaneous
administration of a specific inhibitor of p38 MAPK, FR-167653.
FR-167653 also inhibited augmented expression of tumor necrosis factor
-, connective tissue growth factor, and apoptosis of lung
cells induced by bleomycin administration. Moreover, daily subcutaneous
administration of FR-167653 (from 1 day before to 14 days after
bleomycin administration) ameliorated pulmonary fibrosis and pulmonary
cachexia induced by bleomycin. These findings demonstrated that p38
MAPK is involved in bleomycin-induced pulmonary fibrosis, and its
inhibitor, FR-167653, may be a feasible therapeutic agent.
phosphorylation of p38 mitogen-activated protein kinase; tumor
necrosis factor-; connective tissue growth factor; antiapoptotic
effect
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INTRODUCTION |
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PULMONARY FIBROSIS, which is a heterogeneous group of conditions, is characterized by alveolitis, comprising an inflammatory cellular infiltration into the alveolar septae with resulting fibrosis. Idiopathic pulmonary fibrosis (IPF), whose etiology is unknown, is one of clinically important types of pulmonary fibrosis, and the effects of IPF are chronic, progressive, and often fatal. Previous clinical investigations suggested that the prognosis of IPF is poor with a median survival of 2-2.8 yr (6, 34). However, the pathophysiology of pulmonary fibrosis has not been fully understood.
A number of investigations about pulmonary fibrosis have indicated that
sustained and augmented expression of some cytokines in the lung are
relevant to recruitment of inflammatory cells and accumulation of
extracellular matrix components followed by remodeling of the lung
architecture (8, 22, 27, 32, 58, 59). In particular,
proinflammatory cytokines such as tumor necrosis factor (TNF)-
(35, 36, 39) and interleukin (IL)-1
(40,
48) were demonstrated to play major roles in the formation of
pneumonitis and pulmonary fibrosis. However, the mechanisms of
production of the proinflammatory cytokines and activation of
intracellular signaling cascades triggered by the proinflammatory cytokines are not completely elucidated in pulmonary fibrosis, even in
the animal model of pulmonary fibrosis induced by bleomycin.
The signal transduction pathways through the mitogen-activated protein
kinases (MAPK) are major pathways by which extracellular stimuli are
transmitted to the intracellular signal. Extracellular stimuli such as
oxidative stress, osmotic stress (30), heat shock
(42), ultraviolet irradiation (21),
ischemia-reperfusion (55), lipopolysaccharide
(18), proinflammatory cytokines (5, 41),
DNA-damaging agents (37), and so on were demonstrated to
phosphorylate and activate p38 MAPK and c-Jun NH2-terminal kinase in various types of cells. A synthesized low-molecular-weight pyrazolotriazine derivative, FR-167653, is a potent suppressor of
TNF- and IL-1
production via specific inhibition of p38 MAPK activity (24, 47, 52). Administration of FR-167653 has
been shown to exert beneficial effects in animal models of disseminated intravascular coagulation (51), endotoxin-induced shock
(52), pulmonary ischemia-reperfusion injury
(23), Helicobacter pylori-induced gastritis
(47), and glomerulonephritis (49). Because
effective therapy has not been established for pulmonary fibrosis,
development of an antifibrotic compound is urgently needed. From this
viewpoint, it is worthwhile to assess the anti-inflammatory and
antifibrotic effects of FR-167653. In this investigation, we
administered FR-167653 to a murine model of bleomycin-induced
pulmonary fibrosis and assessed histopathological changes and effects
on cytokine production.
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MATERIALS AND METHODS |
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Reagents. FR-167653 provided by Fujisawa Pharmaceutical (Osaka, Japan) was dissolved at 2% concentration in 0.5% methylcellulose solution (Nikken, Osaka, Japan). To obtain the dosage of 50, 100, or 150 mg/kg body weight (BW), 2.5, 5, or 7.5 µl/g BW of 2% FR-167653 solution was injected subcutaneously. Bleomycin hydrochloride (Nihonkayaku, Osaka, Japan) was dissolved at the concentration of 0.267 U/ml with sterile 0.9% NaCl (isotonic saline) for intratracheal administration. Injected bleomycin solution volume was 3 µl/g BW to obtain the dosage of 0.8 U/kg BW.
Animals. Six-week-old male ICR mice weighing 28-32 g were purchased from Charles River Japan (Yokohama, Japan) and maintained under specific pathogen-free conditions, 12-h light/dark cycles, and constant temperature with food and water ad libitum at the Osaka University animal facility. Animals were allowed to acclimate in facilities for at least 1 wk before any treatments.
Model of bleomycin-induced pulmonary fibrosis and treatment.
The animals were anesthetized with intraperitoneal injections of
pentobarbital sodium (~1 mg; Dainippon Pharmaceutical, Osaka, Japan).
After recognition of anesthetization, we administered the mice
bleomycin solutions intratracheally as previously described (12). Briefly, bleomycin solution was injected through the
vocal cord into the trachea with a Hamilton syringe. The day of
intratracheal injection with bleomycin was determined as day
0. For the experiments to clarify whether FR-167653 modulates
bleomycin-induced pulmonary fibrosis, mice were divided into three
groups. The first group (the saline + vehicle group) was injected with
saline intratracheally in a volume of 3 µl/g BW and treated with
0.5% methylcellulose solution as a vehicle. The second group (the BLM + vehicle group) was injected with bleomycin intratracheally and
treated with vehicle. The third group (the BLM + FR-167653 group) was
injected with bleomycin intratracheally and treated with FR-167653.
Vehicle and FR-167653 were subcutaneously administered once a day from day 1 to day 14. The numbers of mice in the
respective groups are indicated in the figures.
Western blot analysis.
The mice were killed by inhalation of sevoflurane (Maruishi
Pharmaceutical, Osaka, Japan) on days 1, 4, 7, or
21 of bleomycin-induced pulmonary fibrosis. After tracheal
cannulation by a 24-gauge flexible needle, lungs of the mice were
lavaged three times using 1 ml of sterile saline in each wash.
Bronchoalveolar lavage fluid (BALF) cells (2 × 104
cells for Western blot analysis of p38 MAPK) were washed with phosphate-buffered saline, resuspended with sample buffer (62.5 mM
Tris · HCl, pH 7.4, 2% SDS, 50 mM dithiothreitol, 10%
glycerol, and 0.1% bromphenol blue), and boiled for 10 min. The
samples were separated by SDS-PAGE, and the separated proteins were
transferred to a polyvinylidene fluoride membrane (Immobilon;
Millipore, Bedford, MA). Blots were incubated with rabbit primary
antibodies against phosphorylated p38 MAPK (New England Biolabs,
Beverly, MA), and total p38 MAPK (Santa Cruz Biotechnology, Santa Cruz,
CA) was diluted 1:500 in blocking solution for 1 h at room
temperature after incubation for 1 h at room temperature with goat
anti-rabbit horseradish peroxidase-conjugated secondary antibody
(Bio-Rad, Hercules, CA) at a 1:5,000 dilution. Proteins were visualized after incubation of the blots in Western blot chemiluminescence reagent
(NEN, Boston, MA) according to the manufacturer's protocol and
exposure to Fuji RXU film (Fujifilm, Minamiashigara, Japan). For
Western blot analysis of activating transcription factor (ATF)-2, 6 × 104 BALF cells from the saline + vehicle group,
the BLM + vehicle group, and the BLM + FR-167653 group (FR-167653;
150 mg/kg BW) on day 4 (all groups contain 3 mice) were used
and lysed with lysis buffer (20 mM Tris · HCl, pH 7.5, 150 mM
NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium
pyrophosphate, 1 mM -glycerolphosphate, 1 mM
Na3VO4, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Immunoblottings were performed with
antibodies against phosphorylated ATF-2 and total ATF-2 (New England
Biolabs). The ratio of phosphorylated ATF-2:total ATF-2 was determined
by NIH Image analysis.
ELISA of TNF-.
In addition to the bleomycin intratracheal model, we prepared the model
that was administered bleomycin intravenously (bleomycin iv model).
Mice were divided into five groups (each group contains 3 or 4 mice).
The first group was pretreated with 5 µl/g BW of 0.5%
methylcellulose as a vehicle 2 h before a single intravenous injection of 8 µl/g BW saline (saline + vehicle). The second group was pretreated with 5 µl/g BW of 0.5% methylcellulose 2 h
before a single intravenous injection of 8 µl/g BW of 12.5 U/ml
bleomycin solution in sterile saline (the dose of bleomycin was 100 U/kg BW; BLM + vehicle). The other groups were pretreated with 2.5, 5, and 7.5 µl/g BW of 2% FR-167653 solution 2 h before
administration of 100 U/kg BW bleomycin (BLM + FR-167653; 50, 100, and
150 mg/kg, respectively). Two hours after intravenous injection of
saline or bleomycin, mice in each group were killed and blood was
obtained from the right ventricles. After centrifugation of the blood, the sera were stored at
20°C. Mouse serum levels of TNF-
were measured using the mouse TNF-
ELISA kit (R&D Systems, Minneapolis, MN) with a detection limit of 5.1 pg/ml.
Hydroxyproline analysis.
Total collagen content of the whole lungs was estimated by an assay of
hydroxyproline (HOP) (50). Briefly, whole lungs were obtained 21 days after bleomycin administration and hydrolyzed with 2 ml of 6 N HCl at 105°C for 16 h in sealed glass tubes (Iwaki, Tokyo, Japan). HOP in the hydrolysate was determined colorimetrically at 562 nm with p-dimethylaminobenzaldehyde (Sigma, St.
Louis, MO). HOP (Sigma) concentrations from 0 to 10 µg/ml were used
to construct a standard curve. Values are expressed as micrograms of
HOP per pair of lungs. The effect of FR-167653 on the HOP content in
bleomycin-induced pulmonary fibrosis is indicated as "%reduction," which was calculated by the following method: %reduction = (HOP of the BLM + vehicle group HOP of the BLM + FR-167653
group)/(HOP of the BLM + vehicle group
HOP of the saline + vehicle group).
Histological examination. On day 21 of bleomycin-induced pulmonary fibrosis, 1 ml of 10% buffered formaldehyde was instilled to the lung via an intratracheal cannula, and the whole lungs were excised and fixed in 10% buffered formaldehyde in preparation for histological examination (inflation-fixation method). The fixed lungs were embedded in paraffin, sectioned sagittally, and stained with hematoxylin-eosin. For the quantitative histological analysis of fibrotic changes induced by bleomycin, a numerical fibrotic scale (Ashcroft score) (3) was used. The Ashcroft score was obtained as follows. The severity of the fibrotic changes in each lung section was assessed as a mean score of severity from the observed microscopic fields. More than 40 fields covered each whole lung section, observed at a magnification of ×100 in each field, and the severity of the fibrotic changes in each field was assessed and allotted a score from 0 (normal) to 8 (total fibrosis), using the predetermined scale of severity (numerical fibrotic scale). After examination of the whole fields of the section, the mean of the scores from all fields was taken as the fibrotic score. To prevent observer bias, all histological specimens were randomly numbered and interpreted in a blinded fashion. Each specimen was scored independently by three observers.
DNA nick end labeling of tissue sections. Terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labeling (TUNEL) was done with In Situ Cell Death Detection (Roche, Mannheim, Germany) according to the manufacturer's instructions. With this kit, fluorescein isothiocyanate-labeled nucleotides are incorporated at sites of DNA strand breaks by TdT. TUNEL staining was performed on each section from the paraffin-embedded blocks of lungs harvested on day 9. After validating the signals of the TUNEL stain at a magnification of ×400, almost all fields of each section were studied at a magnification of ×100. In each ×100 field, we counted the number of positive signals and calculated the means of the all numbers per field in the three groups.
Northern blot analysis.
The mice were killed on days 4, 7, 14, or 21.
Total RNA from the whole lung was isolated using Isogen (Nippon Gene,
Tokyo, Japan) according to the manufacturer's instructions. Northern blot analysis was performed as described previously (28).
Briefly, 20 µg of RNA were electrophoresed through 1% agarose gels
containing formaldehyde and transferred to a nylon membrane (Hybond N+;
Amersham, Buckinghamshire, UK). Probes used for hybridization were the
full length of cDNA for mouse TNF- and human transforming growth
factor (TGF)-
(44, 56). The probe for mouse connective
tissue growth factor (mCTGF; Fisp-12) was a product of RT-PCR using the
primer pairs sense 5'-GCCAACCGCAAGATTGGAG-3', antisense
5'-TGTAATGGCAGGCACAGGTC-3'. For the densitometric analysis of the mRNA
expressions of TNF-
and mCTGF, mRNA of whole lungs harvested on
day 4 and day 14 was used, respectively. The
ratios of TNF-
:glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and
mCTGF:GAPDH were determined by NIH Image analysis.
Statistical analysis. All values are shown as means ± SE. The significance of differences among groups was assessed with analysis of variance in conjunction with Fisher's least squares difference test. P < 0.05 was considered significant.
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RESULTS |
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Phosphorylation of p38 MAPK and ATF-2 in BALF cells after
intratracheal administration of bleomycin.
We tested phosphorylation of p38 MAPK and ATF-2, one of the substrates
of p38 MAPK, in BALF cells by intratracheal administration of bleomycin
(Fig. 1). In BALF cells from
no-treatment control mouse, basal level of phosphorylated p38 MAPK
(Fig. 1A, top, no-treat lane) was detected. The
samples of the saline + vehicle group on day 4 and day
7 (saline intratracheal on day 0, vehicle subcutaneous on day 1 to day 3 or day 6)
were also tested, and they had the same intensity as the sample of the
nontreated mouse (data not shown). The level of phosphorylated p38 MAPK
of the BLM + vehicle group was markedly augmented one day after
intratracheal administration of bleomycin (0.8 U/kg BW), and the
augmentation lasted for at least 21 days. Also, bleomycin exposure
augmented phosphorylation of ATF-2 of BALF cells on day 4 (Fig. 1, B and C; P = 0.0068). The samples of the nontreated mouse had the same intensity as phosphorylated ATF-2 of the saline + vehicle group on day 4 (data not shown). Next, we tested the in vivo effect of FR-167653 on phosphorylation of ATF-2. The mice were administered with bleomycin on
day 0, and then 150 mg/kg BW FR-167653 was subcutaneously
injected from day
1 to day 3. The BALF cells
were harvested on day 4 of bleomycin treatment. As shown in
Fig. 1, B and C, phosphorylation of ATF-2 in the
BALF cells was significantly inhibited by the FR-167653 treatment
(P = 0.014). Equal loading was checked using an
antibody against total p38 MAPK and total ATF-2 (Fig. 1A,
bottom; Fig. 1B, bottom).
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Effect of FR-167653 on TNF- expression in sera after intravenous
injection of bleomycin.
We elucidated the effect of FR-167653 on TNF-
expression in sera
after intravenous injection of bleomycin. Vehicle or FR-167653 (50, 100, and 150 mg/kg BW) was subcutaneously administered 2 h before
bleomycin (100 U/kg BW) intravenous injection. Two hours after the
bleomycin injection, serum levels of TNF-
were determined by ELISA
(Fig. 2). In the sera from mice that were
pretreated with vehicle and injected with bleomycin (BLM + vehicle),
significant amounts of TNF-
(75.77 ± 11.13 pg/ml) were
detected. The augmentation of TNF-
expression was significantly
reduced to nearly the basal level by the pretreatment of 100 or 150 mg/kg of FR-167653 (P < 0.0001). In the sera of the
mice instilled with saline instead of bleomycin, less than the
detection limit of TNF-
was detected.
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Effect of FR-167653 on HOP content of bleomycin-induced lung
fibrosis.
The effect of FR-167653 on pulmonary fibrosis 21 days after bleomycin
intratracheal administration (0.8 U/kg BW) was assessed by evaluation
of the HOP content of the whole lungs (Fig.
3). The HOP content of the lungs of the
BLM + vehicle group increased approximately twofold compared with
that of the saline + vehicle group in these experiments. The HOP
contents of the BLM + vehicle group were not significantly different
from the lungs instilled with bleomycin alone (not shown). The
treatment with 50, 100, and 150 mg/kg BW of FR-167653 exerted 31.6, 66.1, and 61.9% reduction of the HOP contents compared with treatment
with the vehicle, respectively (P = 0.0169, P = 0.0005, and P < 0.0001; the BLM + vehicle group vs. the BLM + FR-167653 group, respectively). Subsequent
experiments were examined with the treatment of 150 mg/kg BW of
FR-167653.
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Effect of FR-167653 on histopathological changes of
bleomycin-induced pulmonary fibrosis.
The effect of FR-167653 on bleomycin-induced pulmonary fibrosis on
day 21 was also tested histopathologically. The lung
sections of the BLM + vehicle group showed marked histopathological
changes, such as large fibrous areas, collapsed alveolar spaces,
and traction bronchiectasis in the subpleural and peribronchial
regions (Fig. 4B). The
histopathological characteristics of the BLM + vehicle group were
not significantly different from the lungs instilled with bleomycin
alone (not shown). However, in the lungs from the BLM + FR-167653
group (150 mg/kg BW of FR-167653; Fig. 4C), fibrotic lesions
were observed, but its extent was limited and its intensity was
attenuated compared with the BLM + vehicle group. As shown in Fig.
4A, the control lung instilled with saline (the saline + vehicle group) showed no histopathological change. To confirm the
effect of FR-167653 on the histopathological change of
bleomycin-induced pulmonary fibrosis, the overall grades of the
fibrotic changes of the lungs were obtained by numerical score
(Ashcroft score) on day 21 (Fig. 4D). The score
of the BLM + FR-167653 group (2.62 ± 0.22) was significantly
suppressed compared with the BLM + vehicle group (4.72 ± 0.26;
P = 0.0001).
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TUNEL analysis of the lungs on day 9 of bleomycin-induced pulmonary
fibrosis.
We tested the effect of FR-167653 (150 mg/kg BW) on apoptosis
of lung cells induced by bleomycin instillation. To assess
apoptosis, we performed TUNEL staining on the sections of the
lungs on day 9 of bleomycin-induced pulmonary fibrosis and
searched TUNEL-positive signals at a magnification of ×400. The
signals of TUNEL stain were detected at the inflammatory and the
fibrotic foci more abundantly in the BLM + vehicle group than in the
BLM + FR-167653 group, where only a small number of apoptotic
cells were detected. Figure 5A
shows the control lung of the saline + vehicle group, which also shows
few apoptotic cells. The numbers of the signals in total fields of
the lungs were counted at a magnification of ×100 (Fig. 5). The mean
positive cell number per ×100 field was significantly decreased in the
BLM + FR-167653 group (9.95 ± 2.47) compared with the BLM + vehicle group (27.75 ± 6.83; P = 0.026; Fig.
5D). The saline + vehicle group had few apoptotic cells
(4.56 ± 1.27).
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Effect of FR-167653 on TNF-, mCTGF, and TGF-
mRNA expressions
of the whole lungs of bleomycin-induced pulmonary fibrosis.
To further elucidate the effect of FR-167653 on lung inflammation and
also on fibrosis, we assessed TNF-
, mCTGF, and TGF-
mRNA
expression in the lung (Fig. 6). The
lungs were serially obtained from mice instilled with bleomycin and
treated with vehicle or 150 mg/kg BW of FR-167653. In the lungs of the
BLM + vehicle group, increased TNF-
mRNA expression was detected on
day 4 and day 7 of bleomycin-induced pulmonary
fibrosis, and the augmented expression returned to the basal level by
day 14. However, the augmentation of TNF-
mRNA expression
of the BLM + vehicle group was markedly suppressed by the
FR-167653 treatment on day 4 and day 7. By the
densitometric analysis, TNF-
mRNA expression on day 4 of
the BLM + FR-167653 group was statistically lower than the
BLM + vehicle group (Fig. 6B, P = 0.0055).
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Effect of FR-167653 on weight gain of mice.
To test whether FR-167653 ameliorates pulmonary cachexia of
bleomycin-induced pulmonary fibrosis, we compared the weight gain of
mice in each group (Fig. 7). Before
instillation of bleomycin to a mouse on day 0, each mouse
was weighed. On day 21, the mice were weighed again before
being killed. The difference in BW between day 0 and
day 21 was determined as the increase of BW. In the 3 wk of
the experimental course, mice of the saline + vehicle group gained
11.0 ± 0.57 g BW. The mice of the BLM + vehicle
group were cachexic and gained 3.89 ± 0.93 g BW. However,
the mice of the BLM + FR-167653 group (150 mg/kg BW of FR-167653)
gained significantly more weight (6.50 ± 0.63 g) compared
with the BLM + vehicle group (P = 0.028).
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DISCUSSION |
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In the present study, we confirmed that bleomycin exposure induces
p38 MAPK activation and phosphorylation of ATF-2, a substrate of
p38 MAPK, in murine BALF cells, and that the specific inhibitor, FR-167653, inhibited phosphorylation of ATF-2. Moreover, FR-167653 exerted in vivo anti-inflammatory, antifibrotic, and antiapoptotic effects in the mouse model of pulmonary fibrosis induced by bleomycin. These observations strongly suggest that p38 MAPK played a critical role in the pathogenesis of lung fibrosis induced by bleomycin, and the
favorable effect of FR-167653 may be relevant to suppression of TNF-
and CTGF expression. Another important characteristic was that
FR-167653 suppressed the fibrosis formation induced by bleomycin
without any marked adverse effects. These observations suggest that p38
MAPK is an important target of regulation of pulmonary fibrosis, and
FR-167653 may be a feasible novel therapeutic agent.
p38 MAPK was demonstrated to be activated by various extracellular stimuli, and the present study revealed that bleomycin also activates p38 MAPK. After bleomycin exposure, p38 MAPK was phosphorylated as early as day 1, and this phosphorylation sustained to day 21. ATF-2 was also phosphorylated by bleomycin exposure. In BALF cells harvested from mice of the BLM + FR-167653 group on day 4, lower amounts of phosphorylated ATF-2 were detected compared with the BLM + vehicle group (2, 9).
The assays of HOP contents indicated a dose-dependent effect of FR-167653 on bleomycin-induced pulmonary fibrosis. This result revealed the important role of p38 MAPK on bleomycin-induced pulmonary fibrosis. However, the inhibitory effect on fibrosis formation at doses of 100 and 150 mg/kg BW did not differ and reduced the ceiling of ~60% reduction. It is natural that p38 MAPK is one of the signal transducers to contribute to the formation of bleomycin-induced pulmonary fibrosis. Research for other pathways except p38 MAPK in bleomycin-induced pulmonary fibrosis is needed.
In the pathogenesis of pulmonary fibrosis, TNF- augments synthesis
of fibronectin (4), prostaglandin (15), and
TGF-
(38) in vitro. We also observed that the IL-10
gene transfer ameliorated bleomycin-induced pulmonary fibrosis with
suppression of TNF-
expression (1). It was also shown
that in vivo overexpression of TNF-
in the lung induces pulmonary
fibrosis. Sime et al. (45) introduced the TNF-
gene by
the replication-deficient adenovirus vector into rat lungs, and they
observed 7-10 day persistence of the gene expression. Miyazaki et
al. (29) also showed that lung-specific expression of
TNF-
in transgenic mice results in the development of chronic
inflammation and severe fibrosis of the lungs. In the present study,
TNF-
was detected in the sera of the mice 2 h after intravenous
injection of bleomycin, and FR-167653 suppressed serum TNF-
concentration. The present finding regarding the immediate induction of
TNF-
is compatible with previous observations
(46), and the present findings clearly demonstrate that
induction of TNF-
by intravenous administration of bleomycin was
mediated by activation of p38 MAPK. Moreover, we observed significant
suppression of TNF-
mRNA expression in the bleomycin-induced
lung fibrosis model by FR-167653. Some previous studies demonstrated
that another p38 MAPK inhibitor, SB-203580, reduced
lipopolysaccharide-stimulated secretion of TNF-
protein in human
monocytes or lymphocytes but did not distinguish between transcriptional, posttranscriptional, or translational modes of action
(10, 19, 43).
Profibrotic cytokines such as TGF- (7, 14, 33) and CTGF
(11, 26) also play critical roles in the pathogenesis of
pulmonary fibrosis. As shown in Fig. 6, FR-167653 administration significantly suppressed the expression of mCTGF mRNA during the later
phase (day 14) of bleomycin instillation. This observation offers a partial explanation for the amelioration of fibrotic indices
assessed on day 21.
In the present investigation, FR-167653 did not affect TGF- mRNA
expression. It has been reported that posttranslational activation is
more critical than the amount of TGF-
mRNA. The latent form of
TGF-
interacts with
v
6-integrin (31) or
thrombospondin-1 (53), and activation ensues by removal of
latency-associated peptide from the NH2 terminus of the
latent form (54). Hence, the TGF-
mRNA level may not
necessarily reflect the TGF-
activity, and this finding did not
completely negate the relevance of TGF-
to lung fibrosis induced by
bleomycin. Moreover, because the level of CTGF expression was
demonstrated to be upregulated only by TGF-
(20), the
suppressed CTGF expression suggested that TGF-
activity might be
decreased in the lungs of the BLM + FR-167653 group.
It was reported that, in the lungs after bleomycin administration,
apoptosis of bronchial and alveolar epithelial cells is observed (17, 25). Moreover, it was also reported that
apoptosis of pneumocytes induced by agonistic antibody against
Fas results in lung fibrosis (16). In the present
investigation, FR-167653 administration markedly suppressed
apoptosis of the lung cells. The role of p38 MAPK on
apoptosis of various cell types is controversial, but it is
possible that the suppression of apoptosis by FR-167653 is at
least explained by inhibiting death signals transduced by Fas ligand
and TNF- whose expressions were enhanced by p38 MAPK (57).
As shown in Fig. 7, bleomycin reduced the BW gain by inducing pulmonary cachexia. This toxicity of bleomycin assessed by BW gain was partially reversed by FR-167653 administration. This suggested the efficiency of FR-167653 for pulmonary fibrosis. Most investigations assessing the in vivo anti-inflammatory effects of FR-167653 report favorable effects, including amelioration of mortality without marked side effects. However, Gardiner et al. (13) reported that FR-167653 caused increases in plasma creatine kinase and the lactate dehydrogenase level. The toxicity of FR-167653 for primates should be extensively studied, and assessment of its feasibility as an anti-inflammatory drug is an additional problem to be resolved. An important pharmacological feature of FR-167653 is that the compound does not inhibit cyclooxygenase (COX)-1 and COX-2 activities. COX inhibition causes gastrointestinal side effects. Other specific inhibitors of p38 MAPK with pyridinyl imidazole, SB-203580, and RWJ-67657 have also been reported, and they exert inhibitory activity on COX (47). In this respect, FR-167653 may be superior to SB-203580, which has an inhibitory effect on COX activity.
In conclusion, the present study demonstrates that p38 MAPK inhibitor FR-167653 ameliorates the formation of bleomycin-induced pulmonary fibrosis. The present findings suggest that p38 MAPK is involved in bleomycin-induced pulmonary fibrosis. Accumulation of evidence regarding intracellular signal transduction in pulmonary fibrosis may reveal the mechanism of inflammation accompanied with fibrosis and may help to develop potent drugs for pulmonary fibrosis.
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ACKNOWLEDGEMENTS |
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We thank Fujisawa Pharmaceutical (Osaka, Japan) for kindly providing FR-167653 and Yoko Habe for secretarial assistance.
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FOOTNOTES |
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This work was supported by grants from the Japanese Ministry of Education, Culture, Sports, Science, and Technology and the Japanese Ministry of Health, Labor, and Welfare.
Address for reprint requests and other correspondence: H. Matsuoka, Dept. of Molecular Medicine, Osaka Univ. Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 15, 2002;10.1152/ajplung.00187.2001
Received 29 May 2001; accepted in final form 12 February 2002.
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