1 Physiology Program, Harvard School of Public Health, Boston, Massachusetts 02115; and 2 Pulmonary and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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We have previously reported that interleukin
(IL)-1 causes
-adrenergic hyporesponsiveness in cultured human
airway smooth muscle (HASM) cells by increasing cyclooxygenase (COX)-2
expression. The purpose of this study was to determine whether p38
mitogen-activated protein (MAP) kinase is involved in these events.
IL-1
(2 ng/ml for 15 min) increased p38 phosphorylation fourfold.
The p38 inhibitor SB-203580 (3 µM) decreased IL-1
-induced COX-2 by
70 ± 7% (P < 0.01). SB-203580 had no effect on
PGE2 release in control cells but caused a significant
(70-80%) reduction in PGE2 release in IL-1
-treated
cells. IL-1
increased the binding of nuclear proteins to the
oligonucleotides encoding the consensus sequences for activator protein
(AP)-1 and nuclear factor (NF)-
B, but SB-203580 did not affect this
binding, suggesting that the mechanism of action of p38 was not through
AP-1 or NF-
B activation. The NF-
B inhibitor MG-132 did not alter
IL-1
-induced COX-2 expression, indicating that NF-
B activation is
not required for IL-1
-induced COX-2 expression in HASM cells.
IL-1
attenuated isoproterenol-induced decreases in HASM stiffness as
measured by magnetic twisting cytometry, and SB-203580 abolished this
effect. These results are consistent with the hypothesis that p38 is
involved in the signal transduction pathway through which IL-1
induces COX-2 expression, PGE2 release, and
-adrenergic hyporesponsiveness.
mitogen-activated protein; interleukin-1; human airway smooth
muscle; SB-203580; nuclear factor-
B; activator protein-1; prostaglandin E2; cyclooxygenase-2;
-adrenergic
responsiveness; cytoskeletal mechanics; magnetic twisting cytometry
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INTRODUCTION |
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THE CYCLOOXYGENASE
(COX) enzymes COX-1 and COX-2 convert arachidonic acid (AA) to
prostaglandins (PGs) and thromboxane. COX-1 is constitutively expressed
in most tissues, whereas COX-2 is induced by mitogens
(16), bacterial lipopolysaccharide (15), and
cytokines (4, 31). Several recent reports (21, 22, 31) indicated that interleukin (IL)-1 induces COX-2
expression and markedly increases PGE2 release in cultured
human airway smooth muscle (HASM) cells. The induction of COX-2 by
IL-1
in these cells has important functional consequences because
IL-1
decreases the responsiveness of HASM cells to
-agonists, and
this effect is ablated by COX-2 inhibitors and mimicked by exogenous
PGE2 (21, 22, 37). The mechanistic basis for
this effect of COX-2 has not been established, but our results are
consistent with the hypothesis that the marked increase in
PGE2 that results from the induction of COX-2 by IL-1
leads to increased cAMP formation, phosphorylation of the
-adrenergic receptor by protein kinase A (PKA), and consequent
desensitization of the receptor (22, 37). The signal
transduction pathway leading from IL-1
to the induction of COX-2 in
HASM cells has not been established.
In mammalian cells, at least three different subfamilies of
mitogen-activated protein (MAP) kinases have been described. They include the extracellular signal-regulated kinases (ERKs), c-Jun amino-terminal kinase (JNK), and p38. These kinases are activated by
distinct upstream MAP kinase kinases (MEKs) that recognize and
phosphorylate threonine and tyrosine residues within a tripeptide motif
(Thr-X-Tyr) on MAP kinases that is required for their activation. Once
activated, MAP kinases, in turn, phosphorylate a variety of
intracellular substrates including certain transcription factors (18). IL-1 is known to activate all three MAP kinase
subfamilies in HASM cells (27), and Laporte et al.
(21) have previously reported that activation of
the ERK MAP kinases is required for the IL-1
induction of COX-2 and
consequent
-adrenergic hyporesponsiveness in HASM cells.
The purpose of this study was to determine whether p38 activation
is also required for IL-1-induced COX-2 expression, increased PGE2 release, and
-adrenergic hyporesponsiveness in HASM
cells. To do so, we examined the effect of the highly specific
inhibitors of p38 SB-203580 and SB-202190 (6) on COX-2
expression and PGE2 release induced by IL-1
. We also
examined the effect of SB-203580 on the IL-1
-induced changes in HASM
cell responses to the
-agonist isoproterenol (Iso). Because our
results provided evidence of an important role for p38 in these events,
we sought to determine the role of p38 in the signal transduction
pathway leading from IL-1
to the induction of COX-2. Moore et al.
(26) have previously reported that IL-1
increases
activator protein (AP)-1 and nuclear factor (NF)-
B DNA-binding
activity in HASM cells, consistent with its effects in other cell types
(32). Because the promoter region of the human
COX-2 gene contains NF-
B- and AP-1-like consensus
sequences (1) and because p38 has been reported to be
capable of activating both these transcription factors (35,
44), we examined the effect of IL-1
and SB-203580 on the
binding of nuclear proteins to the oligonucleotides encoding the
consensus sequences for AP-1 and NF-
B using electrophoretic mobility
shift assay (EMSA). We also examined the effect of NF-
B inhibitors
on COX-2 expression induced by IL-1
.
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METHODS |
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Cell culture. Human tracheae were obtained from lung transplant donors in accordance with procedures approved by the University of Pennsylvania (Philadelphia, PA) Committee on Studies Involving Human Beings. Tracheal smooth muscle cells were isolated and placed in culture as previously described (14, 21, 22, 30, 37). The cells were cultured on plastic in Ham's F-12 medium supplemented with 10% fetal bovine serum, 102 U/ml of penicillin, 100 µg/ml of streptomycin, 200 µg/ml of amphotericin B, 12 mM NaOH, 1.7 µM CaCl2, 2 mM L-glutamine, and 25 mM HEPES. The medium was replaced every 3-4 days. The cells were passaged with 0.25% trypsin and 1 mM EDTA every 10-14 days. Cells from the nine different donors studied were used in passages 4-7 in the studies described in Experimental protocol. In all cases, the cells were grown to confluence, and 24 h before use were serum deprived and hormone supplemented with 5.7 µg/ml of insulin and 5 µg/ml of transferrin because these conditions maximize expression of smooth muscle-specific contractile proteins (30).
Experimental protocol.
To demonstrate p38 activation by IL-1, we measured p38
phosphorylation in whole cell lysates using Western blotting. HASM cells from the same passage of the same donor cells were treated with
IL-1
(2 ng/ml) for 0, 5, 10, 15, or 30 min. The medium was removed,
and the cells were washed with PBS and then lysed in 400 µl of
extraction buffer [10 mM Tris · HCl buffer with 50 mM NaCl, 50 mM NaF, 10 mM D-serine, 1 mM EDTA, 1 mM EGTA, 1% sodium dodecyl sulfate, 1% Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride
(PMSF), 5 µg/ml of leupeptin, 1 µg/ml of pepstatin, and 10
2 U/ml of aprotinin]. The cells were scraped off the
flasks, passed through a 25
-gauge needle, and solubilized by
sonication. Western blot analysis with an antibody to phosphorylated p38 (New England Biolabs, Beverly, MA) was performed as described, with
details from the manufacturer's protocol. To determine the specificity
of the p38 inhibitor, we measured IL-1
-induced phosphorylation of
p42/p44 and JNK proteins in cells treated with SB-203580 (3 µM) by
Western blot analysis using antibodies to phosphorylated p42/p44 and
JNK (New England Biolabs).
Nuclear protein extracts and EMSA for NF-B and AP-1.
Nuclear extraction was performed with standard methods (20,
25). Briefly, confluent HASM cells were harvested by scraping and centrifugation (3,000 rpm for 5 min) at 4°C in PBS containing a
protease inhibitor cocktail (1 µg/ml of aprotinin, 1 µg/ml of leupeptin, 10 µg/ml of soybean trypsin inhibitor, and 1 µg/ml of
pepstatin). The pellet was washed twice with 1 ml ice-cold buffer
A [10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT), and 0.5 mM PMSF] and recentrifuged as
described above. The supernatant was removed, and the nuclei were
isolated by treating the pellet for 5 min on ice with 60 µl of
buffer A that also contained 0.1% Nonidet P-40 followed by
centrifugation at 14,000 rpm for 10 min at 4°C. The crude pellet was
resuspended in 10 µl of buffer containing HEPES (20 mM, pH 7.9),
MgCl2 (1.5 mM), NaCl (0.42 M), EDTA (0.2 mM, pH 8.0),
glycerol (25% vol/vol), DTT (0.5 mM), and PMSF (0.5 mM) for 15 min on
ice and clarified by centrifugation at 14,000 rpm for 10 min at 4°C.
This supernatant containing the nuclear protein extract was
subsequently diluted with 10 µl of buffer containing HEPES (20 mM, pH
7.9), KCl (50 mM), glycerol (20% vol/vol), DTT (0.5 mM), and PMSF (0.5 mM). Protein concentrations were determined by the bicinchoninic acid system. The nuclear extracts were stored at
70°C.
Transfection of HASM cells.
HASM cells were grown in complete medium for 72 h (60-80%
confluence) in six-well tissue culture plates. Before transfection, the
medium was changed to 1% fetal bovine serum. HASM cells were transfected with 0.5 µg of pNF-B-Luc, designed for monitoring the
NF-
B signal transduction pathway (Clontech, Palo Alto, CA), and 0.5 µg of a
-galactosidase control vector to normalize for differences
in transfection efficiency from well to well. The NF-
B and
-galactosidase vectors were cotransfected with Fugene 6 (Roche,
Indianapolis, IN) according to the manufacturer's protocol. Twenty-four hours posttransfection, the medium was replaced with serum-free medium containing insulin and transferrin as described in
Cell culture. The cells were then incubated with 25 µM
MG-132 or 10 µM PDTC for 17 h and either 0 or 2 ng/ml of IL-1
for 15 h. The cells were lysed with reporter lysis buffer
(Promega, Madison, WI) and harvested. The samples were assayed for
luciferase activity by scintillation counting and for
-galactosidase
activity by spectrophotometry with the
-galactosidase enzyme assay
system (Promega). The results of the experiments are reported as mean luciferase activity normalized to
-galactosidase activity. With this
system, the transfection efficiency averaged 16.4 ± 2.8% (n = 5 experiments) as assessed by flow
cytometry of cells transfected with a green fluorescence
protein-expressing vector.
Magnetic twisting cytometry. Details of the magnetic twisting cytometry technique have been previously described (14, 21, 22, 37, 40, 41). Briefly, ferromagnetic beads (~4 µm in diameter) were coated Arg-Gly-Asp-containing peptides (Peptite 2000, Telios Pharmaceuticals, San Diego, CA) and allowed to bind to adherent HASM cells through integrins that recognize the Arg-Gly-Asp sequence. Individual wells containing bead-coated cells were placed inside the magnetic twisting cytometer and maintained at 37°C with a circulating water bath that is built into the system. The beads were first magnetized with a brief 1,000-G pulse oriented parallel to the surface on which the cells were plated. The magnetic field vector generated by the beads in this direction was measured by an in-line magnetometer. Subsequently, a much smaller magnetic field was applied orthogonally to the first, causing the beads to rotate to align their magnetic moments with this field. This applied torque, or twisting stress (80 dyn/cm2 in this case), is opposed by reaction forces developed within the cytoskeleton to which the beads are bound through the integrin molecules. Magnetic twisting cytometry uses the applied twisting stress and resulting angular rotation of the magnetic beads and expresses the ratio as cell stiffness. Bead rotation increases with the strength of the applied twisting field and is inversely proportional to the resistance of the cell to shape distortion.
Reagents.
The tissue culture reagents and drugs used in this study were from
Sigma (St. Louis, MO) with the exception of amphotericin B and the
trypsin-EDTA solution that were from GIBCO BRL (Life Technologies,
Grand Island, NY), IL-1 that was from Genzyme (Cambridge, MA),
SB-203580 that was from Calbiochem-Novabiochem (La Jolla, CA), and
U-0126 that was a gift from Dupont Pharmaceuticals (Wilmington, DE).
SB-203580, SB-202190, U-0126, and MG-132 were dissolved in DMSO at
concentrations such that the concentration of DMSO in the cell wells
never exceeded 0.1%. PDTC was dissolved in water; and DBcAMP was
dissolved at 10
1 M in distilled water, frozen in
aliquots, and diluted appropriately in medium on the day of use. Iso
(10
1 M in distilled water) was made fresh each day.
Because Iso is rapidly oxidized, dilutions of Iso in medium were made
immediately before addition to the cells.
Statistics.
Changes in basal and BK- and AA-stimulated PGE2 release
induced by IL-1 and SB-203580 were examined by ANOVA, with treatment and experimental day as the main effects. The effect of SB-203580 on
the IL-1
-induced changes in cell stiffness responses to Iso was
examined by repeated-measures ANOVA, with treatment (control, IL-1
,
SB-203580, and IL-1
plus SB-203580) and experimental day as the main
effects. Follow-up t-tests were used to determine where the
treatment effect lay. Effects of p38 inhibitor (SB-203580 or SB-202190)
and NF-
B inhibitor (MG-132 or PDTC) treatment on increased COX-2
expression induced by IL-1
and effects of SB-203580 and U-0126 on
the percent changes in IL-1
-induced AP-1 and NF-
B DNA-binding
activity were examined by paired t-tests of optical densitometry measurements. A P value <0.05 was considered significant.
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RESULTS |
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Activity of p38 in whole HASM cell lysates was estimated by
determining the level of phosphorylated p38 with Western
blotting (Fig. 1A).
There was some p38 phosphorylation even in untreated cells, but p38
phosphorylation began to increase within 5 min after the addition of
IL-1 (2 ng/ml), peaking after 15 min. On average, phosphorylation of
p38 increased fourfold 15 min after treatment with IL-1
(2 ng/ml;
n = 4 donors). In contrast, IL-1
did not alter the
expression of p38 per se (Fig. 1B). To ensure that the
effects of SB-203580 described below were not the result of nonspecific
effects on other MAP kinase pathways that have also been shown to be
activated by IL-1
, we examined the effects of SB-203580 (3 µM) on
MEK1/2 and stress-activated protein kinase (SAPK) activity by measuring
its effects on IL-1
-induced phosphorylation of ERK and JNK. Both ERK
and JNK were phosphorylated in the presence of IL-1
, but
phosphorylation was not altered in the presence of SB-203580 (Fig.
1C). Similar results were obtained in cells from three
different donors.
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IL-1 (2 ng/ml for 22 h) induced COX-2 expression in HASM cells
(Fig. 2) as previously reported
(23), and SB-203580 caused a concentration-dependent
inhibition of this response. In cells treated with 3 and 30 µM
SB-203580 2 h before IL-1
, the density of the COX-2 band was
reduced by 70 ± 7 and 90 ± 7%, respectively (P < 0.01; n = 6 donors/group)
compared with cells treated with IL-1
alone (Fig. 2A).
Similar results were obtained with SB-202190 (3 µM), which caused a
40 ± 20% reduction (n = 3 donors) in the density
of the COX-2 band (Fig. 2B). In contrast, SB-203580 did not
alter the expression of p38 (Fig. 1B), indicating that the effect on COX-2 was not a nonspecific effect on protein synthesis.
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We also examined the effect of SB-203580 on the changes in basal
PGE2 release induced by IL-1 (2 ng/ml for 22 h).
Compared with release in the control cells, IL-1
caused a
significant increase (10- to 20-fold) in basal PGE2
release. Laporte et al. (22) have previously reported that
this increased PGE2 release can be completely ascribed to
the induction of COX-2 evoked by IL-1
. SB-203580 (3 µM) had no
effect on the basal PGE2 release in the control cells but
decreased IL-1
-evoked PGE2 release by 80%
(P < 0.001; Fig.
3). The inhibitory effect of
SB-203580 was not due to cytotoxicity because release of lactate
dehydrogenase into the culture medium was not altered by 24 h of
incubation with 30 µM SB-203580 (data not shown).
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To further evaluate the role of p38 in IL-1-induced prostanoid
release, we examined the effect of prior administration of SB-203580 on
BK (1 µM)- and AA (10 µM)-stimulated PGE2 release in
control and IL-1
-stimulated cells. AA-stimulated PGE2
release requires both the COX and PGE2 synthase enzymes but
not phospholipase A2 (PLA2), whereas in the
case of BK-stimulated PGE2 release, PLA2 must
also be activated. In control cells, SB-203580 (3 µM for 24 h)
had no significant effect on either the BK- or AA-evoked PGE2 release (Fig. 3A). The results with AA
indicate that at this concentration, SB-203580 does not have any
nonspecific effect on PGE2 synthase or COX activity. The
results with BK suggest that p38 does not play a role in BK-induced
PLA2 activation, whereas Laporte et al. (21)
have previously reported that ERK is involved in these events. In
contrast to its lack of effect in control cells, SB-203580 (3 µM)
caused a marked and significant decrease in both BK- and AA-induced
PGE2 release in IL-1
-treated cells (Fig. 3B),
consistent with the effect of the p38 inhibitor on COX-2 expression. We
also examined the effect of a very short preincubation with SB-203580
(3 µM) on AA-stimulated PGE2 release in IL-1
-treated
cells. In this case, SB-203580 was added to the cells for only 30 min
before the addition of AA and not throughout the 22-h period of IL-1
pretreatment, thus being unable to influence IL-1
-induced COX-2
expression. Although long-term (24-h) SB-203580 treatment caused an
~75% reduction in AA-stimulated PGE2 release in
IL-1
-treated cells (P < 0.001; Fig. 3),
short-term (30-min) SB-203580 treatment had no significant effect
on AA-stimulated PGE2 release in IL-1
-treated cells
(24.22 ± 2.6 and 18.48 ± 3.1 ng/ml in IL-1
- and
SB-203580 plus IL-1
-treated cells, respectively). These results
suggest that at this concentration (3 µM), SB-203580 does not have
any nonspecific effects on COX-2 activity and support the idea
that at this concentration, the effect of long term SB-203580 treatment
on PGE2 release in IL-1
-treated cells (Fig. 3) is
through effects of the inhibitor on COX-2 expression.
We also examined the effect of IL-1 and SB-203580 on the binding of
nuclear proteins to the oligonucleotides encoding the consensus
sequences for AP-1 and NF-
B with EMSA. Representative blots are
shown in Fig. 4. Compared with binding in
the control cells, IL-1
increased the binding of nuclear proteins to
the DNA consensus sequences for both AP-1 (Fig. 4A) and
NF-
B (Fig. 4B), and SB-203580 (30 µM) significantly
reduced this effect. When quantified by laser densitometry for 6 experimental days, IL-1
caused a significant increase in nuclear
protein binding to the oligonucleotides encoding the consensus
sequences for both AP-1 and NF-
B (P < 0.01;
Fig. 5). For AP-1,
treatment with 30 µM SB-203580 caused a 57% reduction in
IL-1
-induced binding of nuclear proteins (P < 0.05;
Fig. 5A), but a lower concentration of SB-203580 (3 µM)
had no effect even though this concentration did have significant
effects on COX-2 expression and PGE2 release (Fig. 2). For
NF-
B, SB-203580 at 30 µM again caused a significant reduction in
both the higher and lower molecular weight protein-DNA complexes (32 and 34% reduction, respectively; P < 0.05 for both), but again, a lower SB-203580 concentration (3 µM) had no effect (Fig.
5B). Because Laporte et al. (21) have
previously reported that ERK also participates in IL-1
-induced COX-2
expression in these cells and because ERK has been reported to be
capable of evoking both AP-1 and NF-
B activation (2,
43), we also examined the effect of U-0126 (10 µM) in these
experiments. U-0126 is an inhibitor of the MEK1/2 enzyme that activates
ERK (8). Although there was a trend for U-0126 to inhibit
IL-1
-induced binding of nuclear proteins to the oligonucleotides
encoding the consensus sequences for AP-1, the effect was not
significant. However, U-0126 did cause a small but significant
inhibition of nuclear protein binding to the NF-
B consensus
sequences (P < 0.05 for both bands). To ensure that
the effect of U-0126 was not the result of nonspecific effects on the
p38 MAP kinase pathway, we examined the effects of U-0126 (10 µM) on
p38 phosphorylation induced by IL-1
. Phosphorylation of p38 was not
altered in the presence of U-0126 (data not shown).
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To further evaluate the role of NF-B in COX-2 expression induced by
IL-1
, we examined the effect of two inhibitors of NF-
B activation, MG-132 (29) and PDTC (24), on
IL-1
-induced COX-2 expression with Western blotting. There was no
effect of MG-132 (10 or 25 µM) treatment on IL-1
-induced COX-2
expression (Fig. 6A). Similar
results were obtained in cells from three different donors (Fig.
6B). In contrast, PDTC caused a decrease in COX-2 expression. At 10 µM, PDTC caused a 32% reduction in COX-2
expression as assessed by densitometric analysis of experiments in
three different donors (Fig. 6B). To verify the efficacy of
the NF-
B inhibitors used, we transfected HASM cells with a construct
consisting of
B enhancer elements and a luciferase reporter.
Transfected cells were stimulated with IL-1
in the presence and
absence of PDTC or MG-132. Compared with basal luciferase activity
measured in unstimulated cells, IL-1
increased luciferase activity
(normalized by
-galactosidase activity to control for transfection
efficiency) approximately fivefold (Fig. 6C) and
pretreatment with either MG-132 or PDTC before the addition of IL-1
caused a marked reduction in luciferase activity.
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Laporte et al. (22) have previously reported that
COX-2-generated prostanoids are implicated in IL-1-induced
-adrenergic hyporesponsiveness. Because our results indicated that
p38 was implicated in IL-1
-induced COX-2 expression and prostanoid
release, we sought to confirm that p38 is also involved in the
IL-1
-induced
-adrenergic hyporesponsiveness. To do so, we
examined the effect of SB-203580 (3 µM) on the IL-1
-induced
changes in HASM cell stiffness responses to Iso (Fig.
7). Neither SB-203580, IL-1
, nor their
combination had any effect on baseline cell stiffness [126.15 ± 11.6 dyn/cm2 in control cells; 112.73 ± 5.99 dyn/cm2 in IL-1
-treated cells; 117.13 ± 10.54 dyn/cm2 in SB-203580 (3 µM)-treated cells; 127.08 ± 9.75 dyn/cm2 in SB-203580 plus IL-1
-treated cells]. In
control cells, Iso caused a dose-related decrease in cell stiffness
(Fig. 7). Repeated-measures ANOVA indicated a significant effect of
drug treatment (P < 0.0001) on Iso-induced changes in
cell stiffness. Follow-up analysis indicated that the treatment effect
lay in the response to IL-1
(2 ng/ml), which reduced the capacity of
Iso to decrease cell stiffness as previously described (21, 22,
37). SB-203580 on its own had no effect on the responses to Iso
but abolished the effect of IL-1
.
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Shore et al. (37) have previously shown that IL-1
decreases HASM cell stiffness responses to Iso but has no effect on the cell stiffness responses to DBcAMP. To ensure that the effect of
SB-203580 (Fig. 7) was not the result of nonspecific effects of the
drug on the ability of HASM cells to decrease stiffness, we studied its
effect on the responses to DBcAMP. DBcAMP caused a concentration
dose-related decrease in cell stiffness, consistent with previous
reports (21, 22, 37), but neither IL-1
, SB-203580 (30 µM), nor their combination had any effect on these responses (Fig.
8).
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DISCUSSION |
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In this study, we demonstrated that p38 MAP kinase activation is
involved in the IL-1 signaling pathway leading to COX-2 expression
and PGE2 release in HASM cells. IL-1
caused
phosphorylation of p38 (Fig. 1), and selective p38 MAP kinase
inhibitors (SB-203580 and SB-202190) decreased IL-1
-induced COX-2
expression (Fig. 2) and PGE2 release (Fig. 3). The role of
p38 is unlikely to involve activation of AP-1 and NF-
B because
SB-203580 at 3 µM, a concentration that substantially decreased
IL-1
-induced COX-2 activation (Fig. 2), had no effect on AP-1 or
NF-
B activation after IL-1
stimulation (Fig. 5). PDTC reduced
COX-2 expression induced by IL-1
, but another class of NF-
B
inhibitors, MG-132, did not block IL-1
-induced COX-2 expression,
suggesting that pathways other than NF-
B activation are involved in
the induction of COX-2 by IL-1
(Fig. 6). SB-203580 also blocked the
effects of IL-1
on the HASM responses to the
-agonist Iso (Fig.
7) without affecting the responses to DBcAMP (Fig. 8), consistent with
the role of COX-2 in these events (22).
Our results indicate that IL-1 causes a marked increase in the level
of phosphorylated p38 MAP kinase (Fig. 1), with a time course
consistent with reports in other cell types (11, 12). In
HASM cells and other airway smooth muscle preparations, p38 MAP kinases
have been also shown to be activated by seven-transmembrane-domain receptor ligands such as endothelin, angiotensin, and BK and by growth
factors such as platelet-derived growth factor (9, 23, 28). In each of the studies cited above, as in this study, there was a small amount of p38 phosphorylation even under basal conditions.
The observation that SB-203580 blocked COX-2 expression and
PGE2 release induced by IL-1 is consistent with reports
in other cell types (13, 15, 34) and suggests that p38 is
involved in these events. However, IL-1
induction of COX-2 likely
requires other signaling pathways as well. For example, Laporte et al. (21) have previously reported that IL-1
increases ERK
(p42/p44) phosphorylation and that inhibitors of ERK phosphorylation
substantially reduce IL-1
-increased PGE2 release and
COX-2 expression in HASM cells. In other cell types, both JNK/SAPK and
ERK signaling pathways have been shown to play a role in COX-2
expression and/or PGE2 production (13, 45).
For example, Guan et al. (12) have shown that
overexpression of the dominant negative form of JNK1 or p54
JNK2/SAPK
reduces COX-2 expression and PGE2 production by mesangial cells.
There is a report (42) that p38 MAP kinase is involved in
the activation of cytosolic PLA2 in other cell types.
However, this does not appear to be the case in HASM cells because in
control cells, SB-203580 had no effect on BK-stimulated
PGE2 release (Fig. 3A). SB-203580 did inhibit
BK-stimulated PGE2 release in IL-1-treated cells, but
this effect is likely to have been the result of inhibition of COX-2
expression rather than of PLA2 activation because the magnitude of the effect on BK-induced PGE2 release, which
requires PLA2 activation, and AA induced PGE2
release, which does not, was similar. In contrast to p38, Laporte et
al. (21) have previously reported that ERK appears to
increase PGE2 release both by effects on the induction of
COX-2 and by effects on PLA2 activation.
The inhibitory effects of SB-203580 on COX-2 expression and
PGE2 release were not the result of cytotoxicity because
lactate dehydrogenase release into the culture medium was not altered by 24 h of incubation even with 30 µM SB-203580 and because we observed no effect of SB-203580 on the expression of another protein, p38. We cannot exclude the possibility that the effect of SB-203580 might be the result of nonspecific effects on enzymes other than p38
MAP kinase. However, another p38 inhibitor, SB-202190, with a somewhat
different chemical structure and hence likely to have different
nonspecific effects, had similar effects to SB-203580 on COX-2
expression (Fig. 2B). In addition, other investigators (6) have shown that SB-203580 does not block activation of other MAP kinases (p42/p44, JNK/SAPK), MAP kinase kinase, protein phosphatase, p90 S6 kinase, PKA, or c-Raf. Our results indicate that
SB-203580 also does not block ERK or JNK phosphorylation in HASM cells
(Fig. 1C). It is theoretically possible that the nonspecific
effects of SB-203580 might have contributed to its effect on
IL-1-induced PGE2 release because SB-203580 has been reported to inhibit COX activity in platelets (5).
However, we do not think that this is likely. First, SB-203580 (3 µM)
had no significant effect on AA-stimulated PGE2 release in
control cells (Fig. 3A). SB-203580 did reduce AA-stimulated
PGE2 release in IL-1
-treated cells (Fig. 3B),
but this effect is likely to have been the result of the effects on
COX-2 expression rather than on activity because when SB-203580 (3 µM) was administered to IL-1
-treated cells too late to influence
COX-2 expression, it had no effect on AA-stimulated PGE2
release. Higher concentrations of SB-203580 may have some inhibitory
effects on COX activity because with 30 µM SB-203580, we did observe
a 48% and significant reduction in AA-induced PGE2 release
even in control HASM cells whether SB-203580 was given for 30 min or
24 h (data not shown). Kalmes et al. (17) have shown
that concentrations of SB-203580 > 10 µM can have other
nonspecific effects, and it is possible that such effects may be
responsible for the effects of SB-203580 at 30 µM but not at 3 µM that we observed on AP-1 and NF-
B activation (Figs. 4 and
5).
To begin to address the signal transduction pathway by which p38
activation leads to COX-2 expression, we examined the effect of
SB-203580 on AP-1 and NF-B activation using EMSA. The promoter region of the COX-2 gene contains putative binding sequences
for both these transcription factors, and both have been implicated in
the induction of COX-2 in other cell systems (19). In
response to IL-1
, we observed an increase in the binding of nuclear
proteins to the oligonucleotides containing the consensus sequences for either AP-1 or NF-
B as previously reported in HASM and other cell
types (26, 32, 35, 36). SB-203580 partially inhibited both
AP-1 and NF-
B activation but only at a high concentration (30 µM)
and not at a concentration (3 µM) at which substantive effects of
SB-203580 on IL-1
-induced COX-2 expression were observed (Figs. 4
and 5), suggesting that p38 is unlikely to contribute to the induction
of COX-2 by inducing activation of AP-1 or NF-
B. In contrast,
results with the MEK inhibitor U-0126 (10 µM) at concentrations that
Laporte et al. (21) have previously shown to strongly
inhibit COX-2 expression suggest that ERK appears to contribute only to
the activation of NF-
B, although there was a nonsignificant trend
for U-0126 to inhibit AP-1 DNA-binding activity as well.
NF-B is normally inactive and kept sequestered in the cytoplasm by
its interaction with the inhibitory subunit I
B (3). On
cell activation, I
B is rapidly phosphorylated, ubiquitinated, and
then degraded, resulting in the release and subsequent nuclear translocation of active NF-
B (3). Although the
mechanism by which MAP kinases participate in the regulation of NF-
B
activation is still not firmly established, it has been reported than
MAP kinases or kinases downstream from them may be involved in the phosphorylation of I
B (2). Consistent with these
results, we observed that ERK inhibition caused a small but significant reduction in NF-
B activation in HASM cells. Similarly, Reddy et al.
(32) reported than ERK participates in IL-1
-stimulated NF-
B activation in a human epidermal cell line. In contrast, and
consistent with our results, inhibition of p38 does not appear to
influence NF-
B translocation in a kidney cell line stimulated with
tumor necrosis factor-
and hydrogen peroxide (43). It is possible that in HASM cells, the effects of p38 on COX-2 expression are mediated not at the transcriptional but at the posttranscriptional level. Several studies (7, 33) have reported that
SB-203580 caused a rapid degradation of COX-2 mRNA in cells treated
with IL-1
or lipopolysaccharide, suggesting a role for p38 MAP
kinase in the stability of COX-2 mRNA. The 3'-untranslated region of COX-2 contains 22 AUUUA motifs that are recognized to be important determinants of mRNA instability (7).
Other investigators (10, 39) have reported that in other
cells, the induction of COX-2 can be blocked by NF-B blockers such
as the proteasome inhibitor MG-132 and the oxidant scavenger PDTC. In
our study, we found that MG-132 did not prevent the induction of COX-2
by IL-1
even though PDTC did. We do not know why we found different
effects with the two inhibitors. The lack of effect of MG-132 is not
the result of lack of efficacy because we found that both MG-132 and
PDTC markedly reduced IL-1
-induced NF-
B activity as measured by a
NF-
B reporter assay. The effect of PDTC is not the result of cell
toxicity because we found no effect of PDTC on trypan blue dye
exclusion. Instead it is likely that the NF-
B does not participate
in IL-1
-induced COX-2 expression in these HASM cells because MG-132
did not inhibit IL-1
-induced COX-2 expression even though it
markedly reduced NF-
B activation. The ability of PDTC to inhibit
COX-2 expression may reflect a role for oxidants rather than a role for
NF-
B in these events because the primary activity of the compound is
as an oxidant scavenger (24). The reason for the reported
efficacy of MG-132 against COX-2 expression in another study
(10) but not in this one may be related to differences in
the cell type or the stimulus used to induce COX-2.
Laporte et al. (22) have previously reported that the
mechanism by which IL-1 causes decreased HASM cell responses to
-agonist involves COX-2-generated prostanoid release. In particular,
they showed that exogenous PGE2 mimics the effects of
IL-1
, whereas inhibitors of COX (NS-398 or indomethacin) block the
effects of IL-1
. Our results were consistent with the hypothesis
that the marked increases in basal PGE2 release that result
from the induction of COX-2 by IL-1
lead to increased basal cAMP,
consequent PKA activation, and subsequent phosphorylation and
heterologous desensitization of the
-adrenergic receptor (22,
37). Because the results of this study indicated that p38
activation was required for IL-1
-induced PGE2 release
and COX-2 expression, we reasoned that p38 activation should also be
involved in IL-1
-induced
-adrenergic desensitization. Our results
support that hypothesis. We demonstrated that a selective p38 inhibitor
(SB-203580) inhibited the effects of IL-1
on cell stiffness changes
induced by Iso. These results are consistent with the effect of
SB-203580 on basal PGE2 release: SB-203580 (3 µM) caused
a 70% inhibition in COX-2 expression and an 80% reduction in
PGE2 release.
Cytoskeleton stiffness as measured here is an index of the ability of
cells to resist distortions of shape in response to shear stress
applied through magnetic beads linked to the cytoskeletal network via
integrin receptors. Theoretical modeling studies of such networks
indicate that increasing the interconnectedness of the members, as
would occur during actomyosin interactions, increases the stiffness of
the network (38). Indeed, application of a variety of
contractile agonists to smooth muscle cells results in increased
stiffness, whereas a bronchodilating agonist reduces stiffness
(14, 37). However, changes in cell adhesion can also
influence cytoskeleton stiffness (14, 37), and we cannot rule out the possibility that the observed effects of SB-203580 might
be the result of some role for p38 in cell adhesion. However, we
believe that such an explanation is very unlikely. First, changes in
cell adhesion influence basal cell stiffness in these experiments (14, 37), but neither IL-1, SB-203580, nor the
combination of these agents altered baseline stiffness. Second, such
changes would have been expected to alter cell stiffness response to
any dilating agonist, but the responses to DBcAMP were unaffected by
IL-1
, SB-203580, or their combination (Fig. 8).
In summary, our results indicate that IL-1 activates p38 MAP kinase
and that activation of p38 leads to COX-2 expression and an increase in
PGE2 release but that the role of p38 is unlikely to
involve activation of the transcription factors NF-
B or AP-1. Our
results also indicate that p38 MAP kinase activation is required for
IL-1
-induced
-adrenergic hyporesponsiveness. Understanding the
role of MAP kinases in the mechanism by which cytokines lead to
-adrenergic receptor dysfunction may provide new information for
pharmacological intervention for asthma.
![]() |
ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge the help of Drs. Geoff Maksym and Ben Fabry in maintaining the magnetometer used in the magnetic twisting cytometer experiments.
![]() |
FOOTNOTES |
---|
This study was supported by National Heart, Lung, and Blood Institute Grants HL-56383, HL-33009, HL-55301, and HL-64063 and National Institute of Allergy and Infectious Diseases Grant AI-40203.
J. D. Laporte was the recipient of an American Lung Association fellowship.
Address for reprint requests and other correspondence: S. A. Shore, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115.
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.
Received 19 October 1999; accepted in final form 5 June 2000.
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