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 cells by increasing
cyclooxygenase-2 (COX-2) expression and prostanoid formation. The
purpose of this study was to determine whether extracellular
signal-regulated kinases (ERKs) are involved in these events. Levels of
phosphorylated ERK (p42 and p44) increased 8.3- and 13-fold,
respectively, 15 min after treatment with IL-1
(20 ng/ml) alone.
Pretreating cells with the mitogen-activated protein kinase kinase
inhibitor PD-98059 or U-126 (2 h before IL-1
treatment) decreased
ERK phosphorylation. IL-1
(20 ng/ml for 22 h) alone caused a marked
induction of COX-2 and increased basal
PGE2 release 28-fold
(P < 0.001). PD-98059 (100 µM) and U-126 (10 µM) each decreased COX-2 expression when administered before IL-1
treatment. In control cells, PD-98059 and U-126 had no
effect on basal or arachidonic acid (AA; 10 µM)-stimulated PGE2 release, but both inhibitors
caused a significant decrease in bradykinin (BK; 1 µM)-stimulated
PGE2 release, consistent with a
role for ERK in the activation of phospholipase
A2 by BK. In IL-1
-treated
cells, prior administration of PD-98059 caused 81, 92 and 40%
decreases in basal and BK- and AA-stimulated
PGE2 release, respectively
(P < 0.01), whereas administration
of PD-98059 20 h after IL-1
resulted in only 38 and 43% decreases
in basal and BK-stimulated PGE2
release, respectively (P < 0.02) and
had no effect on AA-stimulated
PGE2 release. IL-1
attenuated
isoproterenol-induced decreases in human airway smooth muscle stiffness
as measured by magnetic twisting cytometry, and PD-98059 or U-126
abolished this effect in a concentration-dependent manner. These
results are consistent with the hypothesis that ERKs are involved early in the signal transduction pathway through which IL-1
induces PGE2 synthesis and
-adrenergic
hyporesponsiveness and that ERKs act by inducing COX-2 and activating
phospholipase A2.
extracellular signal-regulated kinase; mitogen-activated protein; interleukin-1; prostaglandin
E2;
-adrenergic responses; PD-98059; U-126; magnetic twisting cytometry; cyclooxygenase
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INTRODUCTION |
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-ADRENERGIC HYPORESPONSIVENESS is a
characteristic feature of asthma. Decreased bronchodilator responses to
-agonists have been observed in asthmatic airways both in vivo and
in vitro as well as in animal models of asthma (2, 3). There is reason to believe that cytokines may contribute to the
-adrenergic
hyporesponsiveness of asthma. Cytokines such as interleukin (IL)-1
and tumor necrosis factor-
are increased in bronchoalveolar lavage
fluid from symptomatic asthmatic patients (29, 41). In addition, both
IL-1
and tumor necrosis factor-
have been shown to decrease
-adrenergic responsiveness of a variety of cells and tissues
including those in the airways (11, 16, 23, 25, 37, 46).
Cyclooxygenase (COX) activity is the rate-limiting step for the
conversion of arachidonic acid (AA) to prostaglandins (PGs) and
thromboxane (Tx). COX exists in two isoforms. COX-1 is expressed constitutively in most cells (39), whereas COX-2 is induced by mitogens
(19), bacterial lipopolysaccharide (27), and cytokines (4, 32, 39).
Laporte et al. (25) have recently reported that the
mechanistic basis for IL-1-induced decreases in the responsiveness
of cultured human airway smooth muscle (HASM) cells to
-agonists
involves COX-2-induced prostanoid formation. In particular, Laporte et
al. showed that IL-1
leads to COX-2 expression in HASM cells and
that this results in a marked (>10-fold) increase in
PGE2 synthesis. We also
demonstrated that exogenous administration of
PGE2 decreases the responses of
HASM cells to the
-agonist isoproterenol (Iso), whereas COX-2
inhibitors prevent IL-1
-induced
-adrenergic hyporesponsiveness.
Responses to Iso were assessed by measuring changes in cytoskeletal
stiffness by magnetic twisting cytometry (17, 25, 37, 42, 43). Using
this technique, Hubmayr et al. (17) have previously shown
that HASM cells decrease their stiffness in response to any of a panel
of bronchodilator agonists known to cause relaxation of airway smooth
muscle, whereas stiffness increases in response to contractile agonists
known to increase cytosolic calcium concentration in these cells.
Similar results are obtained with vascular smooth muscle cells (26). Although we do not know the precise mechanism by which prostanoids generated in response to IL-1
lead to
-adrenergic
hyporesponsiveness in HASM cells, our results are consistent with the
hypothesis that marked increases in
PGE2 lead to phosphorylation and
heterologous desensitization of the
-adrenergic receptor (25,
37).
The signal transduction pathway leading to COX-2 expression in
IL-1-stimulated airway smooth muscle cells has not been described. However, IL-1
activates a family of protein kinases known as the
mitogen-activated protein (MAP) kinases (14, 15, 18, 35, 38). In other
cell types, MAP kinases appear to be involved in COX-2 induction by
cytokines, lipopolysaccharide, or growth factors (13, 30, 47). In
mammalian cells, at least three subgroups of MAP kinases have been
described, including extracellular signal-regulated kinase (ERK), c-Jun
amino-terminal kinase (JNK), and p38. Two isoforms of ERK, p44 (ERK1)
and p42 (ERK2), are expressed in most cell types and are equally
active. ERK1 and ERK2 require dual phosphorylation for activation (33).
The immediate upstream protein kinase that phosphorylates ERK1 and ERK2
is MAP kinase kinase (MEK) (36). The MAP kinase cascade is one of the
major signaling pathways leading from activation of growth factor,
hormone, or cytokine receptors to induction of genes via their ability to phosphorylate important transcription factors (8, 21, 22). MAP
kinases also phosphorylate other regulatory proteins. For example, ERK
activation induces AA metabolism and the formation of prostaglandins by
its phosphorylation of cytosolic phospholipase A2
(PLA2) (9).
The purpose of this study was to determine whether ERK activation is
required for IL-1-induced prostanoid formation and consequent
-adrenergic hyporesponsiveness in HASM cells. To confirm that IL-1
stimulates ERK in airway smooth muscle and to verify the efficacy of the inhibitors used, we measured ERK phosphorylation in
whole cell lysates. We next examined the effect of two highly specific
inhibitors of MEK phosphorylation and activation, PD-98059 (10) and
U-126 (12), on IL-1
-induced release of
PGE2 and IL-1
-induced COX-2
expression. Finally, we examined the effect of PD-98059 and U-126 on
IL-1
-induced changes in HASM cell responses to the
-agonist Iso.
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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) Committee on Studies Involving Human Beings. A segment of trachea just proximal to the carina was dissected under sterile conditions, and the trachealis muscle was isolated (31). Approximately 1 g of wet trachealis muscle was obtained from each donor. This tissue was minced; centrifuged; resuspended in 10 ml of buffer containing 0.2 mM CaCl2, 640 U of collagenase, 10 mg of soybean trypsin inhibitor, and 10 U/ml of elastase; and incubated with enzymes for 90 min in a shaking water bath at 37°C. The cell suspension so generated was filtered through 127-µm Nytex mesh, and the filtrate was washed with an equal volume of cold Ham's F-12 medium supplemented with 10% fetal bovine serum. For culture, the cells were plated in plastic flasks at 104 cells/cm2 in Ham's F-12 medium supplemented with 10% fetal bovine serum, 102 U/ml of penicillin, 0.1 mg/ml of streptomycin, 2.5 mg/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. Confluent cells were serum deprived and supplemented with 5.7 µg/ml of insulin and 5 µg/ml of transferrin 24 h before use. Cells in passages 4-7 from 13 different donors were used in the studies described below.
Western blotting for measurements of phosphorylated
ERK and COX-2 expression. We measured MEK1/MEK2
activity of whole cell lysates by determining the level of
phosphorylated ERK by Western blotting with an antibody to
phosphorylated p42/p44. COX-2 expression was also measured by Western
blot. In both cases, confluent HASM cells were serum deprived and
treated with PD-98059 (30-100 µM for 15 min or 2 h) or
U-126 (10 µM for 15 min or 2 h) and/or IL-1 (20 ng/ml). IL-1
treatment was for 15 min in the case of p42/p44 activation and for 20 h
in the case of COX-2 expression. 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 (SDS), 1% Triton X-100, 0.2 mM
phenylmethylsulfonyl fluoride, 5 µg/ml of leupeptin, 1 µg/ml of
pepstatin, and 10
2 U/ml of
aprotinin]. Cells were scraped off the flasks, passed through a
25
-gauge needle, and solubilized by sonication.
For p42/p44 Western blots, supernatants of cell lysates were mixed with equal volumes of loading buffer [0.062 M Tris · HCl (pH 6.8), 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, and 0.01% (wt /vol) bromphenol blue] and then were boiled for 5 min. Solubilized proteins (60 µg/lane for p42/p44 and phospho-p42/p44) were separated by SDS-polyacrylamide gel electrophoresis on a 12% Tris-glycine gel (Novex, San Diego, CA) under nonreducing conditions and transferred electrophoretically to a nitrocellulose membrane in transfer buffer (Pierce, Rockford, IL). For p42/p44 and phospho-p42/p44 Western blots, the membrane was blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 for 3 h at room temperature. The blots were probed with rabbit anti-phospho-p42/p44 ERK or anti-p42/p44 ERK antibody (New England Biolabs, Beverly, MA). The phospho-specific antibody recognizes ERK only when phosphorylated at Thr202 and Tyr204. The blots were washed and subsequently incubated (1 h) in Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat dry milk with horseradish peroxidase-conjugated goat anti-rabbit IgG for 2 h. The proteins were visualized by light emission on film with enhanced chemiluminescent substrate (Pierce). The band visualized at ~42 and 44 kDa was quantified with a laser densitometer. Band density values are expressed in arbitrary optical density units. Western blotting for COX-2 was performed as previously described (25).
PGE2 release.
For these experiments, four flasks of HASM cells from the same passage
of the same donor cells were grown to confluence and serum deprived.
Ten hours later, two were treated with PD-98059 (30-100 µM) or
U-126 (10 µM) and the others served as controls. Two hours later,
IL-1 (20 ng/ml) was added to all flasks. Approximately 22 h later,
the cell medium was removed, and the cells were washed with PBS. HASM
cells were harvested by a brief exposure to 0.25% trypsin and 1 mM
EDTA and resuspended in serum-free medium with or without MEK
inhibitors and/or IL-1
. The cells were then plated at
105 cells/well in 24-well plates.
The cells were incubated for 4 h, after which time the medium was
replaced with 0.5 ml of fresh medium. The cells were either left
untreated or AA (10
5 M) or
bradykinin (BK; 10
6 M) was
added. After a 15-min incubation at 37°C, the supernatants were
harvested and stored at
20°C until subsequent assay with a
PGE2 enzyme immunoassay kit
(Cayman Chemical, Ann Arbor, MI). The antibody to
PGE2 had <1% cross-reactivity
to 6-keto-PGF1
and <0.01% to
TxB2 and other PGs according to
the manufacturer's specifications.
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RESULTS |
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MEK1/MEK2 activity of whole HASM cell lysates was estimated by
determining the level of phosphorylated ERK1 and ERK2 (Fig. 1) with Western blotting. ERK1 and ERK2
phosphorylation began to increase within 5 min of addition of IL-1
to HASM cells, peaked after 15 min, and decreased thereafter. To
determine the inhibiting potential of PD-98059 and U-126, two
synthetic, cell-permeable, noncompetitive inhibitors of MEK1/MEK2
phosphorylation and activation, we measured the effect of PD-98059 and
U-126 on IL-1
-induced phosphorylation of ERK1 and ERK2. Compared
with control lysates, IL-1
(20 ng/ml for 15 min) increased the level
of phosphorylation of ERK1 and ERK2 13- and 8.4-fold, respectively, as
assessed by densitometry (n = 3 donors). Pretreatment with 100 µM PD-98059 or 10 µM
U-126 for 2 h before addition of IL-1
decreased IL-1
-increased ERK1 and ERK2 phosphorylation (Fig. 2) but
did not alter ERK expression (data not shown). ERK1 phosphorylation was
inhibited by 88%, whereas ERK2 phosphorylation was inhibited by 77%
in cells treated with PD-98059 (100 µM) compared with that in
IL-1
-treated cells. PD-98059 (30 µM) decreased the level of
phosphorylated ERK1 by 71% and the level of phosphorylated ERK2 by
62%. Pretreatment with U-126 (10 µM) completely abolished
phosphorylation of ERK1 and ERK2 in IL-1
-treated cells. Fifteen
minutes of pretreatment with the MEK1/MEK2 inhibitors also reduced
IL-1
-increased ERK1 and ERK2 phosphorylation (data not shown).
ERK1/ERK2 phosphorylation induced by IL-1
was reduced by 50% in
cells treated with PD-98059 (100 µM) and by 90% in cells treated
with U-126 (10 µM).
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IL-1 (20 ng/ml for 22 h) resulted in marked COX-2 expression (Fig.
3) as previously described (25). Treatment
with PD-98059 (100 µM) for 24 h reduced COX-2 expression (Fig. 3).
Similar results were obtained with U-126. In cells from four different
donors, PD-98059 and U-126 caused a 66 ± 13 and 71 ± 8%
reduction, respectively, in the density of the COX-2 band
(P < 0.02). In contrast, neither PD-98059 nor U-126 had any effect on ERK2 protein expression.
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We also examined the effect of PD-98059 and U-126 on
PGE2 release induced by IL-1
(20 ng/ml for 22 h). Compared with control cells, IL-1
caused a
significant, 28-fold increase in basal
PGE2 release
(P < 0.001) as previously described
(25). Most of this increased PGE2
release results from COX-2 activity (25). PD-98059 (30 or 100 µM)
administered 2 h before IL-1
reduced basal
PGE2 release by 65 (P < 0.001) and 81%
(P < 0.01), respectively. The inhibitory effects of PD-98059 were not due to cytotoxicity because the
release of lactate dehydrogenase into the culture medium was not
altered by 24 h of incubation with 100 µM PD-98059 (data not shown).
U-126 (10 µM) administered 2 h before IL-1
reduced basal PGE2 release by 92%
(P < 0.05).
To further evaluate the role of MEK inhibition in IL-1-induced
prostanoid release, we measured the effect of prior administration of
MEK inhibitors (100 µM PD-98059 or 10 µM U-126 for 24 h) on BK
(10
6 M)- and AA
(10
5 M)-stimulated
PGE2 release in control and
IL-1
(20 ng/ml for 22 h)-stimulated cells (Figs.
4 and
5). AA-stimulated
PGE2 release requires both the COX
and PGE2 synthase enzymes but not
PLA2, whereas in the case of
BK-stimulated PGE2 release,
PLA2 must also be activated.
Both BK and AA caused significant increases in
PGE2 release in control and
IL-1
-pretreated cells compared with basal release (Figs. 4 and 5).
In control cells, PD-98059 (100 µM for 24 h) and U-126 (10 µM for
24 h) caused a marked and significant reduction in BK-stimulated
PGE2 release, consistent with a
report (10) that ERK is involved in BK activation of
PLA2. In contrast, PD-98059
and U-126 had no significant effect on AA-stimulated PGE2 release (Figs.
4A and
5A). In IL-1
-treated cells (Figs. 4B and
5B), PD-98059 and U-126
significantly reduced both BK- and AA-stimulated
PGE2 release, but the effect on
BK-stimulated release was greater than on AA-stimulated release.
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To examine the extent to which effects of ERK on COX-2 expression
versus PLA2 activation contributed
to IL-1-induced PGE2 release,
we also examined the effect of a very short preincubation with PD-98059
(100 µM) on BK- or AA-stimulated
PGE2 release in IL-1
-treated cells. In this case, PD-98059 (100 µM) was
added to the cells for only 15 min before the addition of BK or
AA and not throughout the 22-h period of IL-1
pretreatment, thus
being unable to influence COX-2 expression. Short-term (15-min)
PD-98059 treatment still caused a significant reduction in basal and
BK-stimulated PGE2 release in
IL-1
-treated cells (P < 0.02),
although the magnitude of the effect was not as great as with the 24-h
pretreatment (Fig. 6). In
contrast, although long-term (24-h) PD-98059 treatment caused an
~50% reduction in AA-stimulated
PGE2 release in IL-1
-treated cells (Fig. 4), short-term (15-min) PD-98059 treatment had no significant effect on AA-stimulated
PGE2 release in IL-1
-treated cells (Fig. 6).
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Laporte et al. (25) have previously reported that
prostanoids are implicated in IL-1-induced decreases in HASM cell
responses to
-agonists. Because our data indicated that the ERK MAP
kinases were involved in IL-1
-induced COX-2 expression and
prostanoid release, we sought to determine whether ERK is also involved
in IL-1
-induced
-adrenergic hyporesponsiveness. To do so, we
examined the effect of PD-98059 (100 µM for 24 h) on IL-1
-induced
changes in HASM cell stiffness responses to Iso. The results are shown in Fig.
7A.
Neither PD-98059, IL-1
, nor their combination had any effect on
baseline cell stiffness (129.3 ± 16.0 dyn/cm2 in control cells, 123.0 ± 11.4 dyn/cm2 in
IL-1
-treated cells, 116.0 ± 13.0 dyn/cm2 in PD-98059-treated cells,
and 115.2 ± 7.3 dyn/cm2 in
PD-98059 plus IL-1
-treated cells). In control cells, Iso caused a
dose-related decrease in cell stiffness (Fig.
7A). Repeated-measures ANOVA
indicated a significant effect of drug treatment on Iso-induced changes
in cell stiffness (P < 0.01).
Follow-up analysis indicated that the treatment effect lay in the
response to IL-1
(20 ng/ml), which reduced the capacity of Iso to
decrease cell stiffness as previously described (25, 37). Compared with
control treatment, PD-98059 (100 µM) alone had no effect on the cell
stiffness responses to Iso. However, PD-98059 (100 µM) abolished the
effects of IL-1
on cell stiffness responses to any concentration of
Iso. A lower concentration of PD-98059 (30 µM) did not abolish the
IL-1
response but did significantly reduce the effect of IL-1
at
10
7 and
10
6 M Iso (Fig.
7B). A still lower concentration of
PD-98059 (10 µM) was without effect (data not shown). Similar results
were obtained with U-126 (Fig. 8). Neither U-126, IL-1
, nor their combination had any effect on baseline cell stiffness (114.4 ± 10.9 dyn/cm2 in control cells, 119.0 ± 10.1 dyn/cm2 in
IL-1
-treated cells, 122.5 ± 9.57 dyn/cm2 in PD-98059-treated cells,
and 119.4 ± 10.5 dyn/cm2 in
PD-98059 plus IL-1
-treated cells). Compared with control treatment,
U-126 (10 µM) alone had no effect on the cell stiffness response to
Iso. However, U-126 abolished the effect of IL-1
on the cell
stiffness responses to any concentration of Iso (Fig. 8).
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Shore et al. (37) have previously reported that IL-1
decreases HASM cell stiffness responses to Iso but has no effect on the
cell stiffness responses to dibutyryl cAMP, suggesting that the effect
of IL-1
lies upstream from PKA activation. To ensure that the
effects of PD-98059 (Fig. 9) were
not the result of nonspecific effects of the drug on the ability of
HASM cells to decrease cell stiffness, we also examined the effect of
PD-98059 and IL-1
on the cell stiffness responses to dibutyryl cAMP.
Dibutyryl cAMP induced a concentration-related decrease in cell
stiffness. Neither IL-1
, PD-98059 (100 µM), nor their combination
had any effect on the cell stiffness responses to dibutyryl cAMP (Fig.
9). Furthermore, there was no significant effect of U-126 on the cell
stiffness responses to dibutyryl cAMP in HASM cells (data not shown).
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DISCUSSION |
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Our results indicate that the addition of IL-1 to airway smooth
muscle cells increased phosphorylation of ERK1 and ERK2 (Fig. 1).
Pretreatment of HASM cells with the selective MEK1/MEK2 inhibitors PD-98059 and U-126 reduced this increased phosphorylation (Fig. 2).
IL-1
significantly increased COX-2 expression and
PGE2 release, and the MEK1/MEK2
inhibitors significantly inhibited these events (Figs. 3-6).
PD-98059 and U-126 also blocked the effects of IL-1
on the HASM cell
stiffness responses to the
-agonist Iso (Figs. 7 and 8) without
affecting the responses to dibutyryl cAMP (Fig. 9). Taken together,
these results support the hypothesis that ERK1/ERK2 activation is
involved in IL-1
-induced prostanoid release and
-adrenergic
hyporesponsiveness in HASM cells.
Our results indicate that IL-1 causes a marked increase in the level
of phosphorylated ERK1/ERK2 in HASM cells (Fig. 1). Other investigators
(13, 14) have reported that IL-1
activates p42 and p44 MAP kinases
in other cell types, with a time course similar to that reported here.
In HASM and other airway smooth muscle cells, the ERK MAP kinases have
also been shown to be activated by seven-transmembrane-domain receptor
ligands and by growth factors such as platelet-derived growth factor,
epidermal growth factor, and insulin growth factor I (5, 8, 22, 44).
PD-98059 caused a marked inhibition of IL-1
-induced ERK
phosphorylation in these HASM cells (Fig. 2) at concentrations similar
to those reported as being effective in other cell types (18, 44). Nevertheless, even 100 µM PD-98059 did not completely block the IL-1
-induced phosphorylation of ERK1/ERK2 in these cells. We also
used a second MEK1/MEK2 inhibitor, U-126 (10 µM), which completely inhibited the IL-1
-induced ERK1 and ERK2 phosphorylation in HASM cells, consistent with the results of Favata et al. (12) who reported
that U-126 has an ~100-fold higher affinity for MEK enzymes compared
with PD-98059.
Several groups (32, 40), including ours (25), have reported that
IL-1 induces COX-2 expression and increases
PGE2 release in cultured HASM
cells. In this study, we demonstrated that the signal transduction
pathway leading from IL-1
stimulation to COX-2 expression and
increased PGE2 release
includes ERK activation. In IL-1
-treated cells, we observed a
partial inhibition of COX-2 expression (Fig. 3) and a marked reduction
in PGE2 release (Figs. 4 and
5) in cells treated with the MEK1/MEK2 inhibitors PD-98059 and
U-126. ERK has also been shown to be involved in COX-2 expression induced by lipopolysaccharide in a rat macrophage cell line and in
human monocytes treated with PD-98059 (18, 30). ERK is also
involved in COX-2 expression or
PGE2 release induced by the TxA2 analog U-46619 or by
fibroblast growth factor in porcine aortic smooth muscle cells
(20). The observation that PD-98059 and U-126 did not completely
block the expression of COX-2 induced by IL-1
(Fig. 3) is consistent
with reports by other investigators (18) and suggests that other
pathways may also be implicated in IL-1
-induced COX-2 expression.
For example, the p38 and JNK MAP kinases are also activated by
IL-1
, and in other cell types, p38 inhibitors can block
IL-1
-induced COX-2 expression (13, 38). It is possible that the
inability of PD-98059 to completely block IL-1
-induced COX-2
expression is related to the fact that the inhibitor did not completely
suppress ERK phosphorylation. However, ERK phosphorylation was
virtually abolished by U-126, whereas this inhibitor also did not
completely abolish COX-2 expression.
We do not know the precise mechanism by which ERK activation leads to COX-2 expression. Once activated, ERK translocates to the nucleus and phosphorylates the transcription factor complex TCF/Elk1 (45). Elk1 and serum response factor (SRF) form a complex and bind to a serum response element in the promoter region of some genes. COX-2 gene does not contain a serum response element in its promoter region (47). However, COX-2 gene does have putative activator protein (AP)-1 sites in its regulatory region. This could be important because it has been shown that ERK-activated ternary complex factor/Elk1 does induce c-Fos transcription (28). c-Fos forms heterodimers with the c-Jun family to form AP-1. Thus ERK activation could lead to COX-2 expression via AP-1 activation. The ERK pathway may also phosphorylate an as yet unidentified transcription factor that also participates in the expression of COX-2.
The inhibitory effects of MEK1/MEK2 inhibitors on COX-2 expression and
PGE2 release were not due to
cytoxicity because lactate dehydrogenase release into the culture
medium was not altered by 24 h of incubation with 100 µM PD-98059 and
because we observed no effect of PD-98059 on the expression of another
protein, ERK2. We cannot exclude the possibility that the effects of
PD-98059 or U-126 might be the result of nonspecific effects on enzymes other than MEK1/MEK2. However, other investigators (1, 12) have
demonstrated that PD-98059 and U-126 at the concentrations used in this
study do not inhibit activation of MKK-4, protein kinase C, cdk2, JNK,
MKK3, or p38. In contrast, PD-98059 has been reported to inhibit COX
activity in platelets (6). Although it is theoretically possible that
such nonspecific effects of PD-98059 might have contributed to its
effects on IL-1-induced PGE2
release in this study, we do not think that this is likely. First,
another MEK inhibitor, U-126, with a different chemical structure, had
effects similar to those of PD-98059 on
PGE2 release. Second, neither
PD-98059 nor U-126 treatment had any significant effect on
AA-stimulated PGE2 release in
control cells, indicating that they did not alter COX-1 activity or
PGE2 synthase activity in HASM
cells (Figs. 4 and 5). PD-98059 and U-126 did reduce AA-stimulated PGE2 release in
IL-1
-treated cells (Figs. 4 and 5), but most of this effect is
likely to have been the result of the effects of the compounds on
COX-2 expression (Fig. 3) rather than on activity because when PD-98059
was administered to IL-1
-treated cells too late to influence COX-2
expression, it had no effect on AA-stimulated PGE2 release (Fig. 6).
The reduction in COX-2 expression caused by PD-98059 and U-126 also
contributed to its ability to decrease basal and BK-stimulated PGE2 release in IL-1-treated
cells (Figs. 4B and
5B). However, the effect of long-term PD-98059 and U-126
treatment on basal and BK-stimulated
PGE2 release was greater than the
effect of the inhibitors on AA-stimulated
PGE2 release (Figs.
4B and
5B). In addition, PD-98059 also
inhibited basal and BK-stimulated
PGE2 release even when the drug
was given only 15 min before the supernatants for
PGE2 analysis (Fig. 6).
Furthermore, PD-98059 and U-126 also inhibited BK-stimulated
PGE2 synthesis in control cells
that do not express COX-2 (Figs. 4A
and 5A). Because AA generation of PGE2 requires only COX and
PGE2 synthase, whereas
BK-stimulated PGE2 release also
requires generation of free AA through
PLA2 activity, the most likely
explanation for these data is that ERK activation is important for
BK-stimulated PLA2 activation. In support of this hypothesis, several studies have shown
that the ERK MAP kinases are required for activation of cytosolic
PLA2 by G protein-coupled
receptors such as the BK receptor in other cells systems (24, 34). For
example, Pyne et al. (34) showed that PD-98059 decreased the
phosphorylation of PLA2 induced by BK and abolished the stimulatory effect of BK on
PGE2 release in guinea pig airway
smooth muscle cells.
Laporte et al. (25) have previously reported that the mechanism by
which IL-1 causes decreased HASM cell responses to
-agonists involves COX-2-generated prostanoid release. In particular, we showed
that exogenous administration of
PGE2 mimics the effects of
IL-1
, whereas the inhibition of COX-2 with either NS-398 or indomethacin blocks the effects of IL-1
. Our results suggested that
marked increases in PGE2 lead to
increased basal cAMP, consequent PKA activation, and subsequent
phosphorylation and heterologous desensitization of the
-adrenergic
receptor (25, 37). Because the results of this study indicated that ERK
activation was required for IL-1
-induced
PGE2 release, we reasoned that ERK
activation should also be involved in IL-1
-induced
-adrenergic
hyporesponsiveness. Our results support that hypothesis. In particular,
we demonstrated that the MEK1/MEK2 inhibitors PD-98059 and U-126 caused
a concentration-dependent inhibition of the effects of IL-1
on
the cell stiffness changes induced by Iso (Figs. 7 and 8). In
particular, PD-98059 (100 µM) virtually abolished the responses to
IL-1
, whereas 30 µM had only a partial effect. These results are
consistent with the effect of PD-98059 on basal
PGE2 release: 100 µM caused an
81% inhibition of basal PGE2
release in IL-1
-treated cells, whereas 30 µM had a smaller effect
(65% inhibition).
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 cytoskeleton via integrin
receptors. Actin and myosin form part of the cytoskeleton, and
cross-bridge formation appears to increase cytoskeletal stiffness because the application of a variety of contractile agonists to smooth
muscle cells results in increased stiffness, whereas bronchodilating agonists reduce stiffness (17, 37). The observation that transfection of NIH/3T3 fibroblasts with a tonically active myosin light chain kinase results in increased myosin phosphorylation and also increases cell stiffness compared with cells transfected with an empty plasmid (7) also supports the idea that actomyosin interactions affect cell
stiffness. Changes in cell adhesion to the extracellular matrix can
also influence cytoskeleton stiffness, and Hubmayr et al. (17) have
previously reported that HASM cells plated on high-density collagen are
more spread and develop more pronounced decreases in cell
stiffness in response to Iso than cells plated on a low-density
collagen matrix. Although it is possible that PD-98059 might have
influenced cell adhesion and consequently cell stiffness
responses to Iso, we believe that such an explanation is very unlikely.
First, changes in cell adhesion influence basal cell stiffness (17,
42), but neither IL-1, MEK1/MEK2 inhibitors, nor their combination
altered the baseline stiffness in these experiments. Second, such
changes would have been expected to alter cell stiffness in response to
any dilating agonist, but the responses to dibutyryl cAMP were
unaffected by IL-1
, PD-98059, or their combination (Fig. 9).
In summary, our results indicate that IL-1 activates ERK and that
ERK activation results in increased
PGE2 expression through effects on
both PLA2 activation and COX-2
expression. Our results also indicate that ERK activation is required
for IL-1
-induced
-adrenergic hyporesponsiveness. The observation
that ERK activation is required for IL-1
-induced
PGE2 formation in conjunction with previous results by Laporte et al. (25) indicating that prostanoid formation is necessary for IL-1
-induced
-adrenergic
hyporesponsiveness suggests that the mechanism by which ERK is involved
in IL-1
-induced
-adrenergic hyporesponsiveness is through its
effects on PGE2 formation.
Understanding the role of MAP kinases in the mechanism by which
cytokines lead to
-adrenergic-receptor dysfunction may provide new
avenues for pharmacological intervention for asthma.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. W. Moeller and J. Heyder for synthesizing the magnetic beads and Andrew Esterhas and Igor Schwartzman for technical assistance. U-126 was a gift from Dupont Pharmaceuticals (Wilmington, DE).
![]() |
FOOTNOTES |
---|
This work was supported by National Heart, Lung, and Blood Institute Grants HL-56383 and HL-33009 and fellowships to J. Laporte from the Canadian Lung Association, the Medical Research Council of Canada, and the American Lung Association.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. Laporte, Physiology Program, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115.
Received 10 February 1999; accepted in final form 11 June 1999.
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