Department of Anatomy and Cell Biology, College of Medicine, University of Iowa, Iowa City, Iowa 52242-1109
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ABSTRACT |
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Surfactant protein A
(SP-A), the major lung surfactant-associated protein, mediates
local defense against pathogens and modulates inflammation in the
alveolus. Tumor necrosis factor (TNF)-, a proinflammatory cytokine,
inhibits SP-A gene expression in lung epithelial cells. Inhibitors of
the phosphatidylinositol 3-kinase pathway, i.e., wortmannin, LY-294002,
and rapamycin, did not block the inhibitory effects of TNF-
on SP-A
mRNA levels. An inhibitor of the p44/42 mitogen-activated protein
kinase (MAPK) pathway, PD-98059, was also ineffective. PD-169316 and
SB-203580, inhibitors of p38 MAPK, blocked the TNF-
-mediated
inhibition of SP-A mRNA levels. TNF-
increased the phosphorylation
of p38 MAPK within 15 min. Anisomycin, an activator of p38 MAPK,
increased p38 MAPK phosphorylation and decreased SP-A mRNA levels in a
dose-dependent manner. Finally, TNF-
increased the phosphorylation
of ATF-2, a transcription factor that is a p38 MAPK substrate. We
conclude that TNF-
downregulates SP-A gene expression in lung
epithelial cells via the p38 MAPK signal transduction pathway.
tumor necrosis factor-; surfactant protein A; H441 cells; p38
mitogen-activated protein kinase
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INTRODUCTION |
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SURFACTANT IS A
LIPOPROTEIN that lowers the air-liquid surface tension in the
alveolus and also functions as a component of local defense mechanisms
in the lung (7, 19). Surfactant protein A (SP-A) is the
most abundant surfactant-associated protein (19). SP-A has
been shown to bind to pulmonary pathogens and increase their
phagocytosis by macrophages (7). SP-A may also modulate
inflammatory responses in the alveolus (15, 22). The time-
and dose-dependent inhibitory effect of tumor necrosis factor (TNF)-
on SP-A protein and mRNA levels was first observed in H441 cells, a
human lung epithelial cell line (40). In further studies,
it was shown that TNF-
causes inhibition of SP-A gene transcription
within 6 h (39). The physiological significance of
the TNF-
-mediated decrease in SP-A gene expression is not known;
however, SP-A may act as a general inhibitor of cytokine-mediated inflammation in the lung (4, 15, 22). In agreement with this hypothesis, lipopolysaccharide causes an increased inflammatory response in SP-A null mice compared with its effect in control, wild-type mice (4). Thus the TNF-
-mediated decrease in
SP-A levels may permit a more robust inflammatory response to occur in
the distal lung.
TNF- is a homotrimer of 17-kDa peptide subunits and is produced by
many different cell types in response to inflammation (1).
TNF-
has a wide spectrum of functions, including promoting cell
proliferation, differentiation, apoptosis or survival, and is
implicated in the pathogenesis of many diseases (1).
TNF-
has been shown to play an important role in several lung
inflammatory conditions, including pulmonary fibrosis, acute
respiratory distress syndrome, septic shock, and chronic lung diseases
in infants (8, 11, 27, 37).
TNF- transduces its cellular signal via a complex of receptors,
adapter proteins, and kinases (1). TNF-
binds to two TNF receptors (TNFR), p55 and p75 (type I and type II TNFR,
respectively), with equal affinity (1). H441 cells
express primarily TNFR type I mRNA (30). Interactions
between ligand-bound TNFR and intracellular adapter proteins lead to
the activation of either c-Jun NH2-terminal kinase (JNK),
p38 mitogen-activated protein kinase (MAPK), or nuclear factor
(NF)-
B in lung epithelial cells (3, 5, 18, 25, 31, 32).
Activation of the transcription factor NF-
B by TNF-
is probably
not involved in the regulation of SP-A gene expression by TNF-
(31). TNF-
also activates protein kinase C in human and
bovine lung epithelial cells (3, 41). In isolated rat type
II cells and human lung epithelial cells, TNF-
increases p44/42 MAPK
activity (5, 25). TNF-
has been shown to inhibit de
novo phosphatidylcholine synthesis in H441 cells via an increase in
ceramide production (35). A less common signaling pathway
for TNF-
described in other cell types involves phosphatidylinositol
3-kinase (PI 3-kinase) (9, 16, 24). However, the signaling
pathways utilized by TNF-
to inhibit SP-A gene expression in
pulmonary epithelial cells remain unknown.
In the present study, we used several inhibitors and activators of key
signaling kinases as tools to identify the mediators of TNF-
inhibition of SP-A gene expression in H441 cells. Our results suggest
that the phosphorylation and activation of p38 MAPK in response to
TNF-
results in decreased levels of SP-A mRNA.
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MATERIALS AND METHODS |
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Cell culture.
Human lung epithelial cells, H441 cells, produce SP-A mRNA and protein
(28). H441 cells were obtained from American Type Culture Collection (Rockville, MD) and maintained in monolayer culture
at 37°C and in an atmosphere of 5% CO2 in RPMI 1640 media that contained 10% fetal bovine serum, 0.25 µg/ml amphotericin B, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were split
1:4 weekly, and media were changed every 3-4 days. For most experiments, subconfluent cultures of H441 cells were incubated in
serum-free media for 24 h before the experiment, pretreated with
signal transduction inhibitors or vehicle in fresh media for 2 h,
and then incubated in the presence or absence of the inhibitors plus
TNF- (0.1-100 ng/ml) or vehicle (0.1% bovine serum albumin in
PBS) for an additional 24 h. To evaluate the time dependence of
protein kinase phosphorylation, serum-deprived cells were incubated in
the presence of either TNF-
or vehicle for 15 min, 30 min, 2 h,
and 16 h.
Reagents.
PD-98059 was purchased from New England Biolabs (Beverly, MA).
LY-294002, rapamycin, and wortmannin were obtained from Sigma Chemical
(St. Louis, MO). Anisomycin and SB-203580 were obtained from BioMol
(Plymouth Meeting, PA), PD-169316 was from Calbiochem (San Diego, CA),
and human recombinant TNF- was from either Calbiochem or Sigma.
Stock solutions of PD-98059, wortmannin, LY-294002, rapamycin,
anisomycin, SB-203580, and PD-169316 were prepared in DMSO as 10 mM, 1 mM, 50 mM, 50 µM, 10 mg/ml, 20 mM, and 10 mM stock solutions,
respectively, aliquoted, and stored at
80°C. Antibodies against
total p44/42 MAPK, p38 MAPK, and stress-activated protein kinase/JNK
were obtained from Santa Cruz (Santa Cruz, CA). Antibodies against
phosphospecific forms of p44/42 MAPK, p38 MAPK, JNK, activating
transcription factor-2 (ATF-2), and c-Jun were purchased from Cell
Signaling (Beverly, MA).
Northern blot analysis.
Control and treated cells were harvested, and total RNA was isolated
and then separated in formamide-containing agarose gels according to
previously described methods (26). A photograph of
the ethidium-stained RNA gel was taken to normalize SP-A mRNA levels to
18S ribosomal RNA levels to correct for loading errors, as previously
described (26). The RNA was transferred to a nylon membrane (Nytran SuPerCharge; 0.45-µm pore, Schleicher and Schuell, Keene, NH) by capillary transfer. A human SP-A cDNA was labeled with
[-32P]dCTP using random primers and hybridized to the
membrane that contained the immobilized RNAs. Membranes were then
washed, dried, and exposed to X-ray film for 3-24 h at
70°C
with an intensifier screen.
Immunoblot analysis.
Control and treated cells were rinsed twice with ice-cold 1× PBS and
then lysed in buffer (10 mM Tris · HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1.5 mM MgCl2, 50 mM NaF, 5 mM sodium pyrophosphate, 0.2 mM sodium orthovanadate, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 10% glycerol, 1% Triton X-100, 0.5% Polydet P-40,
and 1 mM phenylmethylsulfonyl fluoride) for 1 h with shaking at
4°C. The lysed cells were scraped and then centrifuged at 12,000 g for 10 min. The lysate supernatant was aliquoted, boiled
for 5 min with 1× electrophoresis sample buffer (125 mM
Tris · HCl, pH 6.8, 1% 2-mercaptoethanol, 2% SDS, 5%
glycerol, and 0.003% bromphenol blue), and stored at 20°C. Equal
amounts of protein (50 µg) were separated on 10% SDS-polyacrylamide
gels, transferred to nitrocellulose membranes, and then nonspecific
binding was blocked by incubating the membrane in Tris-buffered saline
with 5% nonfat dried milk and 0.1% Tween 20 (33). To
detect total p44/42 MAPK, p38 MAPK, JNK1, or c-Jun proteins, rabbit
polyclonal primary antibodies were utilized at 1:1,000, 1:500, 1:200,
and 1:1,000 dilutions, respectively. Rabbit polyclonal phosphospecific
antibodies were used to detect phosphorylated p44/42 MAPK
(Thr202/Tyr204), p38 MAPK
(Thr180/Tyr182), JNK
(Thr183/Tyr185), ATF-2 (Thr71), and
c-Jun (Ser73) (all used at a dilution of 1:1,000).
Polyclonal anti-rabbit IgG horseradish peroxidase-conjugated antibodies
were used as the secondary antibody (1:2,000). An enhanced
chemiluminescence Western blotting detection system (Amersham Pharmacia
Biotech, Piscataway, NJ) was used to visualize the
immunoreactive bands on X-ray film.
p38 MAPK activity assay. The activity of p38 MAPK was measured using a p38 MAPK assay kit (Cell Signaling) according to the manufacturer's instructions. Control and treated cells were rinsed with PBS and lysed. Phospho-p38 MAPK was then immunoprecipitated from the lysate with an immobilized monoclonal antibody, washed, and used in a kinase reaction with ATP and ATF-2 fusion protein. The products of the kinase reaction were separated on an SDS-polyacrylamide gel and immunoblotted with a phospho-ATF-2 antibody as the primary antibody. Immunoreactive bands were detected as described above.
Cell viability.
To evaluate cell viability in the presence of the signal transduction
inhibitors, cells were pretreated with anisomycin, SB-203580, or
vehicle for 2 h and were then incubated for an additional 24 h in the absence of the reagent. H441 cells were also treated with
PD-169316, LY-294002, wortmannin, rapamycin, PD-98059, TNF-, or
vehicle for 24 h. We then performed trypan blue staining,
phase-contrast microscopy, and cell counting on trypsinized H441 cell
cultures. In some experiments, we also measured the lactate
dehydrogenase content of the media (26).
Quantitation and statistical analysis. Reactive bands detected on X-ray films were scanned using a GS-710 scanner (Bio-Rad, Hercules, CA) and quantitated using Quantity One image analysis software (Bio-Rad). Data from treated conditions were normalized to the control conditions, which were made equal to one. Data are presented as means ± SE or SD. All experiments were performed at least three times unless otherwise stated. One-way analysis of variance followed by Dunnett's test or unpaired Student's t-test were used to evaluate significant differences between the treated and control conditions (39).
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RESULTS |
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The toxicities of all of the inhibitors and effectors used in this
study were evaluated by either light microscopy with trypan blue
staining or by lactate dehydrogenase activity measurements. We
evaluated H441 cells treated with TNF- (20 and 100 ng/ml), SB-203580
(20 µM), anisomycin (5 µg/ml), PD-169316 (1 and 3 µM), LY-294002
(5 µM), wortmannin (200 nM), rapamycin (20 nM), and PD-98059 (10 µM) and compared the cell viability with corresponding untreated
controls. Cell viability was not different from controls for all
effectors and signal transduction inhibitor conditions (data not shown).
TNF- inhibits SP-A gene expression.
In initial experiments, we treated serum-starved H441 cells with
TNF-
(0.1-100 ng/ml) in serum-free media for 24 h. TNF-
significantly inhibited SP-A mRNA in a dose-dependent manner, with a
maximum ~70% inhibition achieved at TNF-
concentrations of
10 ng/ml and a half-maximal inhibition (IC50)
at ~4 ng/ml (Fig. 1). In all
further experiments, TNF-
was used at concentrations of 10-100
ng/ml, i.e., at concentrations that resulted in consistently high
levels of SP-A inhibition.
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TNF- does not inhibit SP-A gene expression via the PI 3-kinase
or p44/42 MAPK signal transduction pathways.
Wortmannin and LY-294002 are structurally and mechanistically different
inhibitors of PI 3-kinase with an IC50 of 1-5 nM and 1.4 µM, respectively (2, 36). Rapamycin inhibits the
mammalian target of rapamycin, a protein kinase upstream from p70 S6
kinase, which is a terminal kinase in the PI 3-kinase pathway with an IC50 of ~0.04-0.4 nM (6).
TNF-
-mediated inhibition of SP-A gene expression was not affected by
any of the inhibitors of the PI 3-kinase signaling pathway, i.e.,
LY-294002, wortmannin, or rapamycin (Fig.
2A). In addition, LY-294002,
wortmannin, and rapamycin had no significant effect on basal SP-A mRNA
levels when added alone (Fig. 2A). We also examined the
effects of an inhibitor of the p44/42 MAPK signaling pathway, PD-98059,
on the TNF-
-mediated decrease in SP-A gene expression. PD-98059
blocks activation of MAPK/extracellular signal-regulated kinase kinase
with an IC50 of 10 µM (10). PD-98059 did not
reverse the TNF-
-mediated inhibition of SP-A mRNA levels (Fig.
2B). However, PD-98059 significantly decreased basal levels
of SP-A mRNA when it was added alone (Fig. 2B).
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Inhibitors of p38 MAPK partially block the TNF--induced
inhibition of SP-A gene expression.
We used two inhibitors of the stress-activated protein kinase p38 MAPK,
i.e., SB-203580 and PD-169316, to evaluate the possible role of this
signaling pathway in TNF-
action in H441 cells (42, 43). Both inhibitors are pyrydinylimidazole compounds and bind to the ATP pocket of p38 MAPK (42). SB-203580 and
PD-169316 specifically inhibit p38 MAPK with an IC50 equal
to 74 nM and 89 nM, respectively (12). Both inhibitors may
block the activity of another stress-activated protein kinase, JNK, at
high concentrations (23, 43). SB-203580 is known to
inhibit the
- and
-isoforms of p38 MAPK (38).
Northern blot analysis demonstrated that SB-203580, added alone, had no
effect on SP-A mRNA levels in control cells. However, SB-203580
partially blocked the TNF-
-induced inhibition of SP-A mRNA levels in
a dose-dependent manner (Fig. 3,
A and B). When added alone, PD-169316 had no
effect on SP-A mRNA levels at any concentrations used (Fig. 3,
C and D). However, PD-169316 restored SP-A mRNA
levels from ~40% of control levels in the TNF-
alone condition to
~75% of control levels in the TNF-
plus PD-169316 condition. The
effect of PD-169316 to block the inhibitory effect of TNF-
was dose
dependent.
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Stress-activated protein kinase inhibitors do not affect
TNF--induced phosphorylation of p38 MAPK and JNK.
TNF-
, added alone, significantly increased the phosphorylation of
p38 MAPK (Fig. 4, A and
C). TNF-
also modestly increased the phosphorylation of
JNK; however, the effect was not significant (Fig. 4, B and
D). In additional experiments, TNF-
at a concentration of
10 ng/ml caused a 2.62 ± 0.77-fold increase (means ± SE,
n = 6, P = 0.062, Student's
t-test) in JNK phosphorylation after a 15-min exposure
compared with a 14.24 ± 3.55-fold increase (means ± SE,
n = 7, P < 0.05, Student's
t-test) in p38 MAPK phosphorylation. SB-203580 or PD-169316,
added alone, caused a small increase in the basal phosphorylation of
p38 MAPK, but these effects were not significant (Fig. 4, A
and C). Neither SB-203580 nor PD-169316 had a significant
effect on the TNF-
-induced phosphorylation of p38 MAPK (Fig. 4,
A and C). SB-203580, added alone, also tended to
increase the phosphorylation of JNK at concentrations <20 µM (Fig.
4B). SB-203580 inhibited the TNF-
-induced phosphorylation of JNK only at the 20-µM concentration (Fig. 4B).
PD-169316, added alone, tended to increase JNK phosphorylation in a
dose-dependent manner (Fig. 4D). PD-169316 did not
significantly affect TNF-
-induced JNK phosphorylation at any
concentration tested (Fig. 4D).
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TNF- increases the phosphorylation of p38 MAPK and JNK in a
time-dependent manner.
We next examined the time course of the TNF-
-mediated increase in
the phosphorylation of MAPK family protein kinases in H441 cells. The
phosphorylation state of the MAPKs was slightly increased by the media
change before the experiment but declined within 30 min to 2 h
(Fig. 5). TNF-
caused a synchronous
increase in the phosphorylation of both p38 MAPK and JNK at the 15-min
time point (Fig. 5, B and C). This increase in
phosphorylation of the stress-activated protein kinases was significant
for p38 MAPK (Student's t-test, P < 0.05, n = 3) but not for JNK (Student's t-test,
P = 0.149, n = 3) compared with
corresponding controls. An effect of TNF-
on the phosphorylation of
these two enzymes was absent by the 30-min time point (Fig. 5,
B and C). In further experiments, we found that
20 ng/ml of TNF-
caused a 3.66 ± 1.38-fold increase
(means ± SE, P = 0.09, n = 5, Student's t-test) in JNK phosphorylation after a 15-min
exposure compared with a 8.18 ± 1.59-fold increase (means ± SE, P < 0.05, n = 6, Student's
t-test) in p38 MAPK phosphorylation. TNF-
did not
increase the phosphorylation of p44/42 MAPK at any time point (Fig.
5A). TNF-
also did not alter the total amount of any of
the three MAPKs present in the H441 cells at any time point evaluated
(data not shown).
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TNF- increases p38 MAPK activity.
Serum-starved H441 cells were treated with TNF-
(10 ng/ml) or
vehicle for 15 min, harvested, and assayed for p38 MAPK activity. Densitometric data from three experiments showed that in the presence of TNF-
, p38 MAPK activity was significantly increased and equal to
5.81 ± 2.15 (arbitrary units, means ± SE, Student's
t-test, P < 0.05, n = 3)
compared with activity in the control conditions that were made equal
to one in each experiment.
An activator of p38 MAPK inhibits SP-A gene expression.
Anisomycin is a protein synthesis inhibitor that activates all four p38
MAPK isoforms (,
,
, and
) and JNK2
and their upstream
kinases as well as p70/85 S6 kinase (14). Activation of
stress-activated protein kinases by anisomycin has been shown to lead
to the phosphorylation of several transcription factors, i.e., c-Jun,
ATF-2, and ternary complex factor, and strongly induces the
transcription of early responding genes (14). Pretreatment of H441 cells with anisomycin significantly increased p38 MAPK phosphorylation, in a dose-dependent manner, to approximately three
times the control levels at the 5-µg/ml concentration (Fig. 6, A and B).
Treatment with anisomycin did not affect the total amount of p38 MAPK
at any concentration tested (data not shown, n = 3).
Anisomycin had essentially no effect on JNK phosphorylation in H441
cells (Fig. 6, A and B). In a parallel
experiment, anisomycin significantly inhibited SP-A gene expression
after a 24-h incubation in a dose-dependent manner, with an
IC50 of ~0.4 µg/ml (Fig. 6, C and
D).
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TNF- increases phosphorylation of transcription factors ATF-2
and c-Jun.
ATF-2 is a transcription factor phosphorylated by both p38 MAPK and JNK
(21). The transcription factor c-Jun is a JNK substrate (21). Because TNF-
caused an increased phosphorylation
of p38 MAPK after 15 min of incubation, we evaluated whether the
increase in the phosphorylation of p38 MAPK resulted in ATF-2
phosphorylation. Because we observed a synchronous but smaller effect
of TNF-
on JNK phosphorylation, we also examined the phosphorylation
state of c-Jun in response to TNF-
. Some cells were incubated with anisomycin (10 µg/ml) for 2 h as a positive control. Figure
7A shows that ATF-2
phosphorylation on Thr71 was increased ~10-fold after 15 min of incubation with TNF-
and that the effect was gone by 30 min
of incubation, a time course similar to the TNF-
-induced increase in
p38 MAPK phosphorylation. As shown in Fig. 7B, the
phosphorylation of c-Jun on Ser73 was increased only
modestly (~1.7-fold) in response to TNF-
at 15 min. Anisomycin
treatment of the cells greatly increased the phosphorylation of ATF-2
and modestly increased the phosphorylation of c-Jun (Ser73;
Fig. 7).
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DISCUSSION |
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TNF- inhibits SP-A gene transcription in H441 cells within
6 h (39). Some progress has been achieved in
understanding the molecular events that accompany TNF-
-mediated
suppression of the SP-A gene. For example, Pryhuber et al.
(31) have shown that TNF-
probably does not inhibit
SP-A gene expression via the activation of NF-
B. However, the
signaling pathways utilized by TNF-
in modulating SP-A gene
expression in pulmonary epithelial cells remain unknown.
To gain insight in the TNF- signal transduction mechanisms in lung
epithelial cells, we performed experiments based in part on the TNF-
signaling pathways previously described in lung epithelial cells and in
other cell types. Other investigators have shown that TNF-
can
activate the PI 3-kinase/Akt pathway in some cell types, for example,
to reduce apoptosis of human endothelial cells, to stimulate
survival of rat adult retinal ganglion cells, to promote protein
synthesis in rat cardiac myocytes, and to interfere with insulin
signaling at the insulin receptor substrate-1 (IRS-1) level in kidney
cells (9, 16, 24, 29). We found that the
TNF-
-induced inhibition of SP-A gene expression was not blocked by
either LY-294002 or wortmannin, inhibitors of PI 3-kinase, or by
rapamycin, an inhibitor of the mammalian target of rapamycin, a protein
kinase that regulates p70 S6 kinase (34). Thus our results
in H441 cells suggest that the PI 3-kinase pathway is not involved in
TNF-
-mediated SP-A gene inhibition in lung epithelial cells.
TNF- has been shown to activate p44/42 MAPK as well as p38 MAPK and
JNK in rat and human lung epithelial cells (3, 5, 18, 25).
In our study, in which we used human lung epithelial cell line H441, we
found that TNF-
does not effect the phosphorylation/activation of
p44/42 MAPK. In agreement with these results, inhibition of the p44/42
MAPK pathway using PD-98059 did not reverse TNF-
inhibition of SP-A
gene expression. Interestingly, PD-98059 added alone decreased the
basal levels of SP-A mRNA, a result that suggests the possible involvement of the p44/42 MAPK pathway in maintaining basal SP-A gene
expression in H441 cells. Our observation of a lack of TNF-
-induced p44/42 MAPK phosphorylation in H441 cells differs from the sustained activation of this kinase in rat type II cells reported by Mallampalli and coworkers (25) and in H292 cells, a human alveolar
epithelial carcinoma cell line, as shown by Chen and coworkers
(5). This discrepancy may be the result of differences in
the cell types studied and in the experimental conditions. However,
based on our results, we conclude that the p44/42 MAPK pathway is
probably not involved in TNF-
-mediated inhibition of SP-A gene
expression in H441 cells.
TNF- rapidly increased the phosphorylation and activity of p38 MAPK.
The activation of p38 MAPK was observed at 15 min with a return to
control levels within 30 min of incubation. When we inhibited p38 MAPK
activity with either SB-203580 or PD-169316, SP-A mRNA levels in the
presence of TNF-
were partially restored to the control levels.
SB-203580 and PD-169316 are selective inhibitors of p38 MAPK
- and
-isoforms and may also inhibit some isoforms of JNK at higher
concentrations (12, 23, 38, 42, 43). It has been shown
that SB-203580 binds to the ATP pocket of p38 MAPK and blocks its
catalytic activity but does not affect p38 Thr180 and
Tyr182 phosphorylation (20, 38, 42). In
agreement with this, we found that SB-203580 and PD-169316 do not
inhibit the TNF-
-induced phosphorylation of p38 MAPK in H441 cells.
Furthermore, we showed that anisomycin, an activator of p38 MAPK,
increased the phosphorylation of p38 MAPK and also caused a dramatic
inhibition of SP-A gene expression. We also showed that the activation
of p38 MAPK is accompanied by a synchronical increase in the
phosphorylation of ATF-2, a transcription factor that is a p38 MAPK
substrate (21). Thus we conclude that p38 MAPK is probably
an important signaling pathway by which TNF-
acts to inhibit SP-A
gene expression.
TNF- modestly increased the phosphorylation of JNK simultaneously
with p38 MAPK; therefore, we questioned whether TNF-
might also act
via this kinase to inhibit SP-A gene expression. There is evidence of
some overlap of targets of the p38 MAPK and JNK signaling pathways
(21). For example, TNF-
-induced activation of p38 MAPK
leads to phosphorylation of transcription factors such as ATF-2 and
Elk-1, whereas activated JNK phosphorylates c-Jun, ATF-2, and Elk-1
(21). Our data suggest that the JNK pathway may
be activated in parallel with the p38 MAPK pathway but that the effects
of TNF-
on JNK phosphorylation are much smaller in magnitude than
the effects on p38 MAPK. In agreement with these results, TNF-
caused only a small increase in the phosphorylation of c-Jun, a
transcription factor phosphorylated solely by JNK, compared with a
large increase in ATF-2 phosphorylation, which can potentially be
mediated by either protein kinase, p38 MAPK or JNK (21).
We conclude that TNF-
probably inhibits SP-A gene expression
primarily via the p38 MAPK signaling pathway and not via the JNK pathway.
Our results agree with data reported by Awasthi and coworkers
(3) that TNF- causes a rapid phosphorylation of p38
MAPK that peaks within 15 min in H441 cells. The authors of this study suggested that p38 MAPK is involved in ceramide signaling. In another
human alveolar epithelial cell line, H292 cells, TNF-
treatment
resulted in the phosphorylation of both stress-activated protein
kinases p38 MAPK and JNK as well as p44/42 MAPK (5). These
authors suggest that MAPKs are activated by TNF-
via an increase in
ceramide formation and that this may contribute to NF-
B-mediated
induction of cyclooxygenase-2 gene expression by TNF-
(5). We found that in H441 cells, TNF-
caused
synchronical phosphorylation of p38 MAPK and JNK within 15 min that
ended by 30 min of exposure, a finding that suggests protein
phosphatase-dependent dephosphorylation of these MAPKs may be involved
in their regulation (13).
The phosphorylation of c-Jun, ATF-2, and Elk-1, along with an induction
of early response gene transcription by MAPKs, contribute to activator
protein-1 (AP-1) activation (21). TNF- increases AP-1
binding to the
-glutamylcysteine synthetase heavy subunit promoter
in human lung epithelial cells (32). The human SP-A gene
promoter may contain DNA elements responsive to transcription factor
complexes such as AP-1, which are controlled by the stress-activated protein kinase pathways. Hoover and coworkers (17) have
suggested that binding of transcription factors to an AP-1 site in the
first intron of the SP-A gene (+318/+324) may mediate the inhibitory effects of phorbol ester on SP-A gene expression. This binding may
inhibit SP-A gene transcription by protein-protein interactions and
effects on a basic transcription complex or by interaction with other
cis-acting elements in the regulatory region of the SP-A
gene (17).
In summary, our data suggest that a stress-activated protein kinase
signal transduction pathway, primarily p38 MAPK, is important in the
TNF- regulation of SP-A gene expression in lung epithelial cells.
Neither the p44/42 MAPK nor the PI 3-kinase signal transduction pathways mediate TNF-
inhibition of SP-A gene expression. Our data
do not completely exclude a possible involvement of JNK in TNF-
action. Additional investigation will be needed to identify downstream
effectors in the p38 MAPK signaling pathway that are activated by
TNF-
in lung epithelial cells and to determine how their interaction
with regulatory sequences in the SP-A gene or with other
trans-acting factors results in an inhibition of SP-A gene transcription.
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ACKNOWLEDGEMENTS |
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The authors thank Jean Gardner for typing the manuscript.
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FOOTNOTES |
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The research was supported by National Institutes of Health Grants HL-50050 and DK-25295.
Address for reprint requests and other correspondence: J. M. Snyder, Dept. of Anatomy and Cell Biology, College of Medicine, Univ. of Iowa, 51 Newton Road, 1-550 BSB, Iowa City, IA 52242-1109 (E-mail: jeanne-snyder{at}uiowa.edu).
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.
10.1152/ajplung.00470.2001
Received 11 December 2001; accepted in final form 20 March 2002.
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aggarwal, B.
Tumour necrosis factors receptor associated signalling molecules and their role in activation of apoptosis, JNK and NF-B.
Ann Rheum Dis
59:
I6-I16,
2000[Medline].
2.
Arcaro, A,
and
Wymann MP.
Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses.
Biochem J
296:
297-301,
1993[ISI][Medline].
3.
Awasthi, S,
Vivekananda J,
Awasthi V,
Smith D,
and
King RJ.
CTP:phosphocholine cytidylyltransferase inhibition by ceramide via PKC-, p38 MAPK, cPLA2, and 5-lipoxygenase.
Am J Physiol Lung Cell Mol Physiol
281:
L108-L118,
2001
4.
Borron, P,
McIntosh JC,
Korfhagen TR,
Whitsett JA,
Taylor J,
and
Wright JR.
Surfactant-associated protein A inhibits LPS-induced cytokine and nitric oxide production in vivo.
Am J Physiol Lung Cell Mol Physiol
278:
L840-L847,
2000
5.
Chen, CC,
Sun YT,
Chen JJ,
and
Chang YJ.
Tumor necrosis factor--induced cyclooxygenase-2 expression via sequential activation of ceramide-dependent mitogen-activated protein kinases, and I
B kinase 1/2 in human alveolar epithelial cells.
Mol Pharmacol
59:
493-500,
2001
6.
Chung, J,
Kuo CJ,
Crabtree GR,
and
Blenis J.
Rapamycin-FKBP specifically blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases.
Cell
69:
1227-1236,
1992[ISI][Medline].
7.
Crouch, E,
and
Wright JR.
Surfactant proteins A and D and pulmonary host defense.
Annu Rev Physiol
63:
521-554,
2001[ISI][Medline].
8.
De Dooy, JJ,
Mahieu LM,
and
Van Bever HP.
The role of inflammation in the development of chronic lung disease in neonates.
Eur J Pediatr
160:
457-463,
2001[ISI][Medline].
9.
Diem, R,
Meyer R,
Weishaupt JH,
and
Bahr M.
Reduction of potassium currents and phosphatidylinositol 3-kinase-dependent AKT phosphorylation by tumor necrosis factor- rescues axotomized retinal ganglion cells from retrograde cell death in vivo.
J Neurosci
21:
2058-2066,
2001
10.
Dudley, DT,
Pang L,
Decker SJ,
Bridges AJ,
and
Saltiel AR.
A synthetic inhibitor of the mitogen-activated protein kinase cascade.
Proc Natl Acad Sci USA
92:
7686-7689,
1995[Abstract].
11.
Fahey, TJ, III,
Tracey KJ,
and
Cerami A.
Tumor necrosis factor (cachectin) and the adult respiratory distress syndrome.
In: Update: Pulmonary Diseases and Disorders, edited by Fishman AP.. New York: McGraw-Hill, 1992, p. 175-183.
12.
Gallagher, TF,
Seibel GL,
Kassis S,
Laydon JT,
Blumenthal MJ,
Lee JC,
Lee D,
Boehm JC,
Fier-Thompson SM,
Abt JW,
Soreson ME,
Smietana JM,
Hall RF,
Garigipati RS,
Bender PE,
Erhard KF,
Krog AJ,
Hofmann GA,
Sheldrake PL,
McDonnell PC,
Kumar S,
Young PR,
and
Adams JL.
Regulation of stress-induced cytokine production by pyridinylimidazoles: inhibition of CSBP kinase.
Bioorg Med Chem
5:
49-64,
1997[ISI][Medline].
13.
Haneda, M,
Sugimoto T,
and
Kikkawa R.
Mitogen-activated protein kinase phosphatase: a negative regulator of the mitogen-activated protein kinase cascade.
Eur J Pharmacol
365:
1-7,
1999[ISI][Medline].
14.
Hazzalin, CA,
Le Panse R,
Cano E,
and
Mahadevan LC.
Anisomycin selectively desensitizes signalling components involved in stress kinase activation and fos and jun induction.
Mol Cell Biol
18:
1844-1854,
1998
15.
Hickman-Davis, JM,
Fang FC,
Nathan C,
Shepherd VL,
Voelker DR,
and
Wright JR.
Lung surfactant and reactive oxygen-nitrogen species: antimicrobial activity and host-pathogen interactions.
Am J Physiol Lung Cell Mol Physiol
281:
L517-L523,
2001
16.
Hiraoka, E,
Kawashima S,
Takahashi T,
Rikitake Y,
Kitamura T,
Ogawa W,
and
Yokoyama M.
TNF- induces protein synthesis through PI3-kinase-Akt/PKB pathway in cardiac myocytes.
Am J Physiol Heart Circ Physiol
280:
H1861-H1868,
2001
17.
Hoover, RR,
Pavlovic J,
and
Floros J.
Induction of AP-1 binding to intron 1 of SP-A1 and SP-A2 is implicated in the phorbol ester inhibition of human SP-A promoter activity.
Exp Lung Res
26:
303-317,
2000[ISI][Medline].
18.
Janssen-Heininger, YM,
Macara I,
and
Mossman BT.
Cooperativity between oxidants and tumor necrosis factor in the activation of nuclear factor (NF)-B: requirement of Ras/mitogen-activated protein kinases in the activation of NF-
B by oxidants.
Am J Respir Cell Mol Biol
20:
942-952,
1999
19.
Johansson, J,
and
Curstedt T.
Molecular structures and interactions of pulmonary surfactant components.
Eur J Biochem
244:
675-693,
1997[Abstract].
20.
Kumar, S,
Jiang MS,
Adams JL,
and
Lee JC.
Pyridinylimidazole compound SB-203580 inhibits the activity but not the activation of p38 mitogen-activated protein kinase.
Biochem Biophys Res Commun
263:
825-831,
1999[ISI][Medline].
21.
Kyriakis, JM,
and
Avruch J.
Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation.
Physiol Rev
81:
807-869,
2001
22.
LeVine, AM,
and
Whitsett JA.
Pulmonary collectins and innate host defense of the lung.
Microbes Infect
3:
161-166,
2001[ISI][Medline].
23.
Liverton, NJ,
Butcher JW,
Claiborne CF,
Claremon DA,
Libby BE,
Nguyen KT,
Pitzenberger SM,
Selnick HG,
Smith GR,
Tebben A,
Vacca JP,
Varga SL,
Agarwal L,
Dancheck K,
Forsyth AJ,
Fletcher DS,
Frantz B,
Hanlon WA,
Harper CF,
Hofsess SJ,
Kostura M,
Lin J,
Luell S,
O'Neill EA,
Orevillo CJ,
Pang M,
Parsons J,
Rolando A,
Sahly Y,
Visco DM,
and
O'Keefe SJ.
Design and synthesis of potent, selective, and orally bioavailable tetrasubstituted imidazole inhibitors of p38 mitogen-activated protein kinase.
J Med Chem
42:
2180-2190,
1999[ISI][Medline].
24.
Madge, LA,
and
Pober JS.
A phosphatidylinositol 3-kinase/Akt pathway, activated by tumor necrosis factor or interleukin-1, inhibits apoptosis but does not activate NF-B in human endothelial cells.
J Biol Chem
275:
15458-15465,
2000
25.
Mallampalli, RK,
Peterson EJ,
Carter AB,
Salome RG,
Mathur SN,
and
Koretzky GA.
TNF- increases ceramide without inducing apoptosis in alveolar type II epithelial cells.
Am J Physiol Lung Cell Mol Physiol
276:
L481-L490,
1999
26.
Metzler, MD,
and
Snyder JM.
Retinoic acid differentially regulates expression of surfactant-associated proteins in human fetal lung.
Endocrinology
133:
1990-1998,
1993[Abstract].
27.
Moller, DR.
Systemic sarcoidosis.
In: Fishman's Pulmonary Diseases and Disorders, edited by Fishman AP.. New York: McGraw-Hill, 1998, p. 1055-1068.
28.
O'Reilly, MA,
Gazdar AF,
Morris RE,
and
Whitsett JA.
Differential effects of glucocorticoid on expression of surfactant proteins in a human lung adenocarcinoma cell line.
Biochim Biophys Acta
970:
194-204,
1988[ISI][Medline].
29.
Ozes, ON,
Akca H,
Mayo LD,
Gustin JA,
Maehama T,
Dixon JE,
and
Donner DB.
A phosphatidylinositol 3-kinase/Akt/mTOR pathway mediates and PTEN antagonizes tumor necrosis factor inhibition of insulin signaling through insulin receptor substrate-1.
Proc Natl Acad Sci USA
98:
4640-4645,
2001
30.
Pryhuber, GS,
Huyck HL,
Staversky RJ,
Finkelstein JN,
and
O'Reilly MA.
Tumor necrosis factor--induced lung cell expression of antiapoptotic genes TRAF1 and cIAP2.
Am J Respir Cell Mol Biol
22:
150-156,
2000
31.
Pryhuber, GS,
Khalak R,
and
Zhao Q.
Regulation of surfactant proteins A and B by TNF- and phorbol ester independent of NF-
B.
Am J Physiol Lung Cell Mol Physiol
274:
L289-L295,
1998
32.
Rahman, I,
Antonicelli F,
and
MacNee W.
Molecular mechanism of the regulation of glutathione synthesis by tumor necrosis factor- and dexamethasone in human alveolar epithelial cells.
J Biol Chem
274:
5088-5096,
1999
33.
Sharma, RV,
Tan E,
Fang S,
Gurjar MV,
and
Bhalla RC.
NOS gene transfer inhibits expression of cell cycle regulatory molecules in vascular smooth muscle cells.
Am J Physiol Heart Circ Physiol
276:
H1450-H1459,
1999
34.
Shepherd, PR,
Withers DJ,
and
Siddle K.
Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling.
Biochem J
333:
471-490,
1998[ISI][Medline].
35.
Vivekananda, J,
Smith D,
and
King RJ.
Sphingomyelin metabolites inhibit sphingomyelin synthase and CTP: phosphocholine cytidylyltransferase.
Am J Physiol Lung Cell Mol Physiol
281:
L98-L107,
2001
36.
Vlahos, CJ,
Matter WF,
Hui KY,
and
Brown RF.
A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002).
J Biol Chem
269:
5241-5248,
1994
37.
White, CW,
and
Das KC.
Role of cytokines in acute lung injury.
In: The Lung: Scientific Foundations, edited by Crystal RG,
West JB,
Weibel ER,
and Barnes PJ.. Philadelphia: Lippincott-Raven, 1997, p. 2451-2464.
38.
Whitmarsh, AJ,
and
Davis RJ.
Analyzing JNK and p38 mitogen-activated protein kinase activity.
Methods Enzymol
332:
319-336,
2001[ISI][Medline].
39.
Whitsett, JA,
Clark JC,
Wispe JR,
and
Pryhuber GS.
Effects of TNF- and phorbol ester on human surfactant protein and MnSOD gene transcription in vitro.
Am J Physiol Lung Cell Mol Physiol
262:
L688-L693,
1992
40.
Wispe, JR,
Clark JC,
Warner BB,
Fajardo D,
Hull WE,
Holtzman RB,
and
Whitsett JA.
Tumor necrosis factor- inhibits expression of pulmonary surfactant protein.
J Clin Invest
86:
1954-1960,
1990[ISI][Medline].
41.
Wyatt, TA,
Ito H,
Veys TJ,
and
Spurzem JR.
Stimulation of protein kinase C activity by tumor necrosis factor- in bovine bronchial epithelial cells.
Am J Physiol Lung Cell Mol Physiol
273:
L1007-L1012,
1997
42.
Young, PR,
McLaughlin MM,
Kumar S,
Kassis S,
Doyle ML,
McNulty D,
Gallagher TF,
Fisher S,
McDonnell PC,
Carr SA,
Huddleston MJ,
Seibel G,
Porter TG,
Livi GP,
Adams JL,
and
Lee JC.
Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase bind in the ATP site.
J Biol Chem
272:
12116-12121,
1997
43.
Zhang, Y,
Zhong S,
Dong Z,
Chen N,
Bode AM,
Ma W,
and
Dong Z.
UVA induces Ser381 phosphorylation of p90RSK/MAPKAP-K1 via ERK and JNK pathways.
J Biol Chem
276:
14572-14580,
2001