1 Physiology Program, Harvard School of Public Health, Boston, Massachusetts 02115; and 2 Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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IL-1 inhibits isoproterenol
(ISO)-induced relaxation of cultured human airway smooth muscle (HASM)
cells. The purpose of this study was to determine whether IL-1
can
also suppress ISO-induced cAMP response element (CRE)-dependent gene
expression. ISO (10 µM) caused a marked increase in CRE-binding
protein (CREB) phosphorylation, which was attenuated by IL-1
(2 ng/ml). This effect of IL-1
was abolished by the cyclooxygenase
(COX) inhibitor indomethacin. To examine CRE-driven gene expression, we
transiently transfected HASM cells with a construct containing CRE
upstream of a luciferase reporter gene. ISO (6 h) caused a sixfold
increase in luciferase activity. IL-1
(24 h) alone also increased
luciferase activity, although to a lesser extent (2-fold). However, the
ability of ISO to elicit luciferase expression was markedly reduced in
cells treated with IL-1
. Indomethacin, the MEK and p38 inhibitors
U-0126 and SB-203580, the protein kinase A inhibitor H-89, and
dexamethasone each completely abolished the ability of IL-1
to
induce CRE-driven gene expression but only slightly increased the
ability of ISO to induce CRE-driven gene expression in IL-1
-treated
cells. IL-1
also attenuated dibutyryl cAMP-induced CRE-driven gene
expression, but not dibutyryl cAMP-induced CREB phosphorylation. Tumor
necrosis factor-
(10 ng/ml) also attenuated ISO-induced CRE-driven
gene expression, even though it was without effect on ISO-induced cAMP formation or ISO-induced CREB phosphorylation. The results suggest that
IL-1
and tumor necrosis factor-
may attenuate the ability of
-agonists to induce expression of genes with CRE in their regulatory
regions at least in part through events downstream of CREB phosphorylation.
mitogen-activated protein kinase; cyclooxygenase-2; dexamethasone; cAMP response element binding; luciferase; tumor necrosis factor-
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INTRODUCTION |
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DECREASED
RESPONSIVENESS to -adrenergic receptor agonists is a
characteristic feature of asthma. Airways of asthmatic patients have a
reduced capacity to dilate in response to
-agonists in vivo
(4) and in vitro (3). Animal models of asthma
also exhibit decreased responses to
-agonists (8). The
mechanistic basis for these changes in responsiveness has not been
established, but it is possible that cytokines released in the
asthmatic airway may act directly on airway smooth muscle cells to
exert these effects. For example, we previously reported that
interleukin (IL)-1
decreases the capacity of cultured human airway
smooth muscle (HASM) cells to generate cAMP and to relax in response to
isoproterenol (ISO) (31). Similar results have been
obtained in other species (11, 35).
ISO exerts its effects in smooth muscle by binding to the
2-adrenergic receptor, which couples to the stimulatory
G protein, Gs, which activates adenylyl cyclase, resulting
in cAMP release. cAMP, in turn, activates protein kinase A (PKA), which
results in cell relaxation through effects on K+ channels,
Na+-K+-ATPases, Ca2+ sequestration,
Ca2+ sensitivity of myosin, and inositol trisphosphate
formation (28, 33). In addition to relaxing HASM cells,
-agonist activation of PKA also induces changes in the transcription
of genes that contain cAMP response elements (CRE) in their promoter
regions (13, 27, 32). For example, the promoters of
several genes that have been shown to be important for the regulation
of smooth muscle cell function, such as the cyclooxygenase (COX)-2 gene (26), the IL-6 gene (1), and the gene for the
2-adrenergic receptor itself (19), contain
CRE elements that are important in inducing gene transcription.
Activation of CRE elements by ISO occurs through PKA phosphorylation of
CRE-binding protein (CREB) at serine-133 (6, 29).
Phosphorylation of CREB permits its interaction with CREB-binding
protein (CBP) and p300, which interact with the basal transcriptional
apparatus to initiate transcription (6).
The purpose of this study was to determine whether IL-1, in addition
to its ability to inhibit
-agonist-induced relaxation of smooth
muscle, also inhibits
-agonist-induced CRE-driven gene expression.
To that end, we examined ISO-induced CREB phosphorylation in the
presence and absence of IL-1
. We also examined the effects of ISO
and IL-1
alone and in combination on luciferase expression in HASM
cells transiently transfected with a luciferase reporter construct
driven by multiple CRE elements. Our results indicate that IL-1
substantially reduces the ability of ISO to evoke CRE-driven gene
expression. Similar results were obtained with a representative gene,
IL-6, which contains a CRE element in its promoter.
We previously reported that although IL-1 decreases the ability of
HASM cells to relax in response to ISO, IL-1
does not affect HASM
cell relaxation in response to dibutryl cAMP (DBcAMP), a cell-permeant
analog of cAMP that directly activates PKA. IL-1
also has no effect
on the ability of forskolin, which directly activates adenylyl cyclase,
to generate cAMP (31). These data support the hypothesis
that the effect of IL-1
on ISO-induced relaxation of HASM cells is
mediated upstream of adenylyl cyclase, i.e., at the level of
-receptor-Gs interactions. Our data also indicated that
ISO-induced HASM relaxation occurs through a COX-2-dependent pathway
(16), because we showed that IL-1
markedly upregulates COX-2 expression and prostaglandin E2 (PGE2)
release, that the effect of IL-1
on ISO-induced cAMP formation is
abolished in cells that are treated with COX-2 inhibitors, and that
exogenous PGE2 mimics the effect of IL-1
. Taken
together, the data are consistent with the hypothesis that
IL-1
-induced PGE2 release results in increased basal
cAMP formation and PKA activation, leading to phosphorylation of the
2-receptor, uncoupling it from Gs. Further
investigations indicated that the effect of IL-1
, which was
dependent on activation of the extracellular signal-regulated kinase
(ERK) and p38 mitogen-activated protein (MAP) kinase pathways acting to
induce COX-2 expression (14, 15), could be blocked with
glucocorticoids (20). For these reasons, we examined the effects of the COX inhibitor indomethacin, the MAP kinase kinase (MEK)
inhibitor U-0126 (9), the p38 inhibitor SB-203580
(5), and the glucocorticoid dexamethasone on ISO-induced
CRE-driven gene expression. We also examined the effect of IL-1
on
DBcAMP-induced CREB phosphorylation and CRE-driven gene expression.
Because tumor necrosis factor-
(TNF-
) is also present in
asthmatic airways and can interact with IL-1
to promote
-adrenergic desensitization (18), we also examined the
effect of TNF-
on ISO-induced CREB phosphorylation and CRE-driven
gene expression.
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METHODS |
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Cell culture. HASM cells were obtained from lung transplant donor tracheas 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. Smooth muscle cells were isolated from the trachealis as previously described (21). For culture, the cells were plated into plastic flasks at 104 cells/cm2 in Ham's F-12 medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (0.1 mg/ml), amphotericin B (2.5 mg/ml), 12 mM NaOH, 1.6 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 deprived of serum and supplemented with insulin (5.7 µg/ml) and transferrin (5 µg/ml) 24 h before use. Cells in passages 4-7 from 18 different donors were used in the studies described below.
Western blotting for measurement of phosphorylated CREB.
HASM cells were grown to confluence in six-well plates and deprived of
serum for 24 h as described above. For measurement of
phosphorylated CREB, cells were treated with ISO (10 µM for 15 min)
and IL-1 (2 ng/ml for 24 h) alone or in combination. Similar
experiments were carried out in cells pretreated with the COX inhibitor
indomethacin (1 µM) 2 h before treatment with IL-1
. After
treatment for the appropriate time, the culture medium was removed and
the cells were washed with PBS and then lysed with 200 µl of
extraction buffer [10 mM Tris · HCl buffer with 50 mM NaCl, 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 leupeptin, 1 µg/ml pepstatin, and 10
2 U/ml
aprotinin]. The cells were scraped from the plates, passed through a
25
Transfection of HASM cells.
After passage, HASM cells were grown in complete medium for 72 h
(60-80% confluence) in six-well plates. Before transfection, the
medium was changed to 1% fetal bovine serum. HASM cells were transfected with 0.5 µg of pCRE-luciferase (pCRE-Luc), designed for
monitoring the CRE 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
pCRE-Luc vector contains three copies of a consensus CRE-binding
sequence fused to a TATA-like promoter region upstream of the gene for
firefly luciferase. Further details about the vector are available on
the manufacturer's website. The CRE and
-galactosidase vectors were
cotransfected with Fugene 6 (Roche, Indianapolis, IN) according to the
manufacturer's protocol. At 24 h after transfection, the medium
was replaced with serum-free medium containing insulin and transferrin
as described above. The cells were then incubated with IL-1
(2 ng/ml
for 24 h) and ISO (10 µM for 6 h) alone or in combination.
The above protocol was also repeated in the presence or absence of the
MEK inhibitor U-0126 (10 µM; Promega, Madison, WI). Inhibition of MEK
prevents the phosphorylation and activation of ERK. We also repeated
the protocol in the presence and absence of the p38 inhibitor SB-203580 (3 µM; Calbiochem-Novabiochem, La Jolla, CA), indomethacin (1 µM),
or dexamethasone (1 µM). Each of these drugs was administered 2 h before addition of IL-1
. We also repeated the protocol using DBcAMP (1 mM) instead of ISO and using TNF-
(10 ng/ml) instead of
IL-1
. The cells were lysed with reporter lysis buffer (Promega) 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. Drugs and
reagents were obtained from Sigma (St. Louis, MO) unless otherwise indicated.
Measurement of cAMP.
Cells were treated with TNF- (10 ng/ml) or left untreated. After
20 h, cells were harvested by brief exposure to trypsin and EDTA,
resuspended in serum-free medium with or without TNF-
, and plated at
105 cells/well in 24-well plates. After the cells were
allowed to readhere for 4 h at 37°C, the medium was replaced
with 0.5 ml of PBS containing 0.1 mM IBMX (to prevent degradation of
cAMP by phosphodiesterases) and 300 µM ascorbic acid (to prevent
oxidation of ISO). After 30 min, ISO (10 µM) or diluent was added to
the cells. Cells were incubated for an additional 10 min and then removed from the incubator and placed on ice. Ice-cold ethanol (1 ml)
was added to lyse the cells. The lysate was centrifuged at 2,000 g for 15 min at 4°C, and the supernatant was removed, evaporated to dryness, and stored at
70°C until assay. On the day
of assay, the cell lysate was resuspended in 200-500 µl of assay
buffer. Some samples were diluted to ensure that they fell within the
limits of the standard curve. cAMP was assayed using a Rainen cAMP
125I radioimmunoassay kit (New England Nuclear).
Statistics. The significance of drug treatment on CREB phosphorylation (densitometry), CRE-driven luciferase expression, and cAMP formation was assessed by ANOVA using drug treatment and experimental day as main effects. Follow-up t-tests were used to assess where the drug treatment effect lay. Bonferroni's rule was used to correct for multiple comparisons. P < 0.05 was considered significant.
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RESULTS |
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ISO-induced CREB phosphorylation.
Figure 1A shows the effect of
ISO (10 µM for 15 min) and IL-1 (2 ng/ml for 24 h) on CREB
phosphorylation. ISO caused a large increase in CREB phosphorylation
compared with control cells. When cells were treated with IL-1
(2 ng/ml) 24 h before addition of ISO, ISO-induced CREB
phosphorylation was largely suppressed (Fig. 1A). Similar
results were obtained in cells from three HASM cell donors, and
densitometric analysis of the data indicated that the decrease in
ISO-induced CREB phosphorylation caused by IL-1
was statistically
significant (P < 0.05; Fig. 1B). In
contrast, IL-1
did not attenuate ISO-induced CREB phosphorylation in
cells that had been treated with indomethacin (1 µM) 2 h before
addition of IL-1
(Fig. 1).
|
ISO-induced CRE-driven gene expression.
HASM cells were transfected with a pCRE-Luc construct and treated with
ISO (10 µM for 6 h) and IL-1 (2 ng/ml for 24 h) alone and in combination. ANOVA indicated a statistically significant effect
of drug treatment (P < 0.001) on CRE-driven luciferase expression (Fig. 2). ISO caused an
approximately sixfold increase (P < 0.001) in
luciferase expression normalized for
-galactosidase expression
compared with untreated cells, whereas IL-1
resulted in an
approximately twofold increase in luciferase expression (P < 0.001). When cells were treated first with
IL-1
and 18 h later with ISO, the ability of ISO to increase
luciferase activity was markedly diminished (P < 0.001). Indeed, the effect of ISO (difference between ISO-treated and
control cells) in IL-1
-treated cells averaged only 12 ± 3% of
the response to ISO in cells not treated with IL-1
.
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DBcAMP-induced CREB phosphorylation and CRE-driven gene expression.
DBcAMP (1 mM for 15 min), like ISO, increased CREB phosphorylation
(Fig. 5). However, in contrast to the
effect of IL-1 on ISO-induced CREB phosphorylation (Fig. 1), IL-1
(2 ng/ml for 24 h) did not alter DBcAMP-induced CREB
phosphorylation (Fig. 5). Nevertheless, IL-1
did reduce
DBcAMP-induced CRE-dependent gene expression (Fig.
6). DBcAMP caused a marked increase in
luciferase expression in untreated cells transfected with a pCRE-Luc
reporter. However, in cells treated with IL-1
, the effect of DBcAMP
was significantly reduced (P < 0.05). Taken together,
the results suggest that the effects of IL-1
on DBcAMP-induced
changes in CRE-driven gene expression must lie downstream of CREB
phosphorylation.
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Effect of TNF- on ISO-induced CREB phosphorylation and
CRE-driven gene expression.
We previously reported that TNF-
has only minimal effects on the
ability of ISO to relax HASM cells (18). To confirm that TNF-
also does not alter ISO-induced changes in cAMP formation, as
has previously been reported (22), we measured changes in cAMP induced by ISO in cells treated with or without TNF-
(10 ng/ml
for 24 h). In contrast to IL-1
, which increases basal cAMP formation (31), TNF-
did not alter basal cAMP formation
in HASM cells (43.3 ± 6.8 vs. 47.6 ± 7.1 pmol/106 cells). ISO (10 µM for 15 min) increased cAMP
formation, but there was no significant difference in the response to
ISO in cells treated with TNF-
compared with control cells
(209.5 ± 22.6 and 208.5 ± 23.7 pmol/106 cells,
respectively). Similarly, TNF-
did not alter ISO-induced CREB
phosphorylation (Fig. 7). In contrast to
IL-1
, TNF-
did not alter basal CRE-driven gene expression in HASM
cells (Fig. 8), likely because TNF-
does not induce COX-2 expression in HASM cells (18).
However, similar to IL-1
, TNF-
did attenuate the ability of ISO
to induce luciferase expression (Fig. 8). Taken together, these results
suggest that the effect of TNF-
on CRE-driven gene expression must
lie downstream of CREB phosphorylation.
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Effect of IL-1 on ISO-induced IL-6 promoter activity.
To confirm the relevance of our results for genes with CRE in their
promoters, we repeated these experiments in cells transfected with
pIL-6-Luc 651 instead of the CRE-Luc construct (Fig.
9). ISO stimulates IL-6 promoter activity
in HASM cells, and this effect of ISO is dependent on the presence of a
CRE at position
154 (2). Our results (Fig.
9A) indicated that ISO caused an 84 ± 34% increase
(P < 0.05) in luciferase in pIL-6-Luc 651-transfected cells but failed to increase IL-6 promoter activity in IL-1
-treated cells. We also repeated the experiments in cells transfected with pIL-6-Luc 651
NF-
B (Fig. 9B) to eliminate effects of
IL-1
on IL-6 promoter activity that were independent of its effects
on the
-adrenergic pathway. Mutation of the NF-
B binding site at position
63 of the IL-6 promoter completely abolished the ability of
IL-1
to stimulate IL-6 promoter activity. ISO still caused a robust
increase in promoter activity in control cells but failed to increase
promoter activity in cells treated with IL-1
. Taken together, the
results indicate that IL-1
also attenuates ISO-induced CRE-driven
IL-6 expression.
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DISCUSSION |
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We previously reported that IL-1 suppresses ISO-induced cAMP
formation and relaxation in HASM cells (31). The results
of the present study demonstrate that IL-1
also inhibits the ability of ISO to induce CREB phosphorylation and CRE-driven gene expression (Figs. 1 and 2). The effect of IL-1
on ISO-induced CREB
phosphorylation appears to require COX-2. In contrast, the effect of
IL-1
on ISO-induced CRE-driven gene expression is only slightly
attenuated by COX-2 inhibitors and appears to result, at least in part,
from effects downstream of CREB phosphorylation, because IL-1
also suppressed DBcAMP-driven gene expression (Fig. 6) without altering DBcAMP-induced CREB phosphorylation (Fig. 5).
We previously reported that although IL-1 causes a marked inhibition
of the ability of ISO to induce cAMP formation and relax airway smooth
muscle, the cytokine has no effect on responses to DBcAMP or forskolin
(31). These results suggest that the effect of IL-1
on
ISO-induced cAMP formation lies upstream of adenylyl cyclase. Because
2-receptor expression and Gs expression were
also unaffected, we reasoned that IL-1
was acting to uncouple the
-adrenergic receptor from Gs (31). We
subsequently demonstrated that the ability of ISO to cause cell
relaxation and to induce cAMP formation was restored by COX-2
inhibitors and mimicked by PGE2 (16). Similar
results have been obtained by other investigators (22).
Because IL-1
by itself caused a small but significant increase in
cAMP formation, we reasoned that IL-1
might be acting by increasing
COX-2 expression and PGE2 release, resulting in increased
cAMP formation. We further reasoned that the increased PKA activity
resulting from this increase in cAMP could be causing the decrease in
-adrenergic responsiveness by phosphorylating the
-receptor
(12, 17, 25), uncoupling it from Gs.
Our results indicate that this mechanism also accounts for the ability
of IL-1 to attenuate ISO-induced CREB phosphorylation, because this
effect was abolished by indomethacin (Fig. 1). In contrast, this
mechanism is likely to account for only a small part of the effect of
IL-1
on ISO-induced CRE-driven gene expression, because the COX
inhibitor indomethacin restored only a small portion of the ability of
ISO to induce CRE-driven luciferase expression (Fig. 4), likely as a
result of its effects on CREB phosphorylation. Furthermore, U-0126 and
SB-203580, inhibitors of the ERK and p38 MAP kinase pathways, and
dexamethasone also reversed only a very small part of the effect of
IL-1
on ISO-induced CRE-driven gene expression (Fig. 4), even though
all these compounds substantially attenuate IL-1
-induced COX-2
expression and completely abolish the loss of ISO-induced relaxation
responses that are observed in IL-1
-treated cells (14, 15,
20). It is unlikely that the inability of these agents to
restore the effects of ISO on CRE-driven gene expression is due to a
lack of efficacy, because we previously reported that, at the
concentrations used, U-0126 virtually abolishes the ability of IL-1
to induce ERK phosphorylation (14), whereas indomethacin,
dexamethasone, and SB-203580 also abolish the ability of IL-1
to
induce PGE2 release in HASM cells (15, 16,
20). Instead, the results suggest that an additional mechanism
accounts for the majority of the effect of IL-1
on ISO-induced
CRE-driven gene expression.
We do not know the precise details of this additional mechanism.
However, the observation that IL-1 does not alter DBcAMP-induced CREB phosphorylation (Fig. 5) but does attenuate CRE-driven gene expression mediated by DBcAMP (Fig. 6) suggests that there must be
effects of IL-1
on events downstream of CREB phosphorylation. Because interaction of CBP and p300 with phosphorylated CREB is vital
to the formation of the basal transcriptional apparatus that allows
transcription of CRE-dependent genes (6), it is possible
that the ability of IL-1
to attenuate ISO- or DBcAMP-induced CRE-driven gene expression is the result of effects on recruitment of
CBP to phosphorylated CREB. CBP interacts not only with CREB, but also
with other transcription factors (6, 10, 29, 30), some of
which, e.g., NF-
B and AP-1, are activated by IL-1
in HASM cells
(20), and it has been hypothesized that nuclear
competition between transcription factors for limiting amounts of CBP
results in mutual repression of the ability of these factors to
activate transcription (30). Alternatively, it has also
been reported that the C/EBP family of transcription factors can
interact with CRE in some genes (24). We have reported
that IL-1
induces C/EBP
expression in HASM cells
(34), and it is possible that this transcription factor
competes with CREB for binding to CRE.
Although IL-1 attenuated ISO-induced CREB phosphorylation (Fig. 1),
our results suggest that this effect played only a minor role in the
reduction in ISO-induced CRE-driven gene expression that was caused by
IL-1
. First, IL-1
also attenuated DBcAMP-induced CRE-driven gene
expression, but this occurred without any effect on DBcAMP-induced CREB
phosphorylation (Figs. 5 and 6). Second, indomethacin completely
restored the ability of ISO to induce CREB phosphorylation in
IL-1
-treated cells (Fig. 1), but even after restoration of CREB
phosphorylation, IL-1
continued to attenuate ISO-induced CRE-driven
gene expression (Fig. 4), albeit not to the same extent as in cells not
treated with indomethacin.
TNF-, like IL-1
, is increased in asthmatic airways. Our results
indicate that TNF-
, like IL-1
, also suppresses CRE-driven gene
expression in HASM cells (Fig. 8). Because TNF-
did not alter
ISO-induced cAMP formation, it is likely that the cytokine must be
exerting its effects downstream of
-receptor-Gs coupling and adenylyl cyclase activation. Because TNF-
does not alter cell
stiffness responses to DBcAMP (18), the cytokine cannot be
affecting PKA activity. Taken together with our results indicating no
effect of TNF-
on ISO-induced CREB phosphorylation (Fig. 7), the
data indicate that the effect of TNF-
on CRE-driven gene expression
(Fig. 8) is also mediated downstream of CREB.
IL-1 induction of COX-2 appears to be only minimally involved
in the effect of IL-1
on ISO-induced CRE-driven gene expression (Fig. 4). However, COX-2 does appear to contribute to the ability of
IL-1
alone to increase CRE-driven gene expression in HASM cells,
because the COX inhibitor indomethacin completely blocked this effect
(Fig. 3). In addition, U-0126 and SB-203580 inhibited the effects of
IL-1
on CRE-driven gene expression, consistent with the requirement
of the p38 and ERK MAP kinases for IL-1
-induced COX-2 expression
(14, 15). Dexamethasone, which blocks IL-1
-induced COX-2 expression (20), was also effective in abolishing
IL-1
-induced CRE-driven gene expression. Hence, it is likely that
IL-1
induces CRE-driven gene expression as follows. IL-1
induces
COX-2, leading to PGE2 release. PGE2 acts on EP
receptors on the smooth muscle cells to induce cAMP formation and
subsequent PKA induction. PKA then phosphorylates CREB, which recruits
CBP, leading to gene expression. These results are consistent with our
previous observations that IL-1
causes a small but significant
increase in basal cAMP formation in HASM cells that can be abolished by
COX-2 inhibitors (16) and that the PKA inhibitor H-89
inhibited the effects of IL-1
on CRE-driven gene expression (Fig.
3).
In summary, our results indicate that ISO-induced CRE-driven gene
expression is suppressed by pretreatment with IL-1 or TNF-
in
HASM cells and that the effect of these cytokines is likely to be at
least partly mediated by events downstream of CREB phosphorylation. These effects of IL-1
may have important implications for the expression of genes such as IL-6, which contain CRE in their regulatory regions. Thus IL-1
, in addition to inhibiting
-agonist-induced HASM cell relaxation, also inhibits the effect of
-agonists on CRE-dependent gene expression, both of which may be important in asthma.
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ACKNOWLEDGEMENTS |
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The authors thank I. Schwartzman and T. Church for excellent technical assistance.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants HL-67664, HL-56383, HL-33009, HL-55301, HL-64063, and AI-40203. P. E. Moore was supported by National Heart, Lung, and Blood Institute Grant HL-04395 and grants from the American Lung Association and the Charles H. Hood Foundation.
Address for reprint requests and other correspondence: S. A. Shore, Physiology Program, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115 (E-mail: sshore{at}hsph.harvard.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.
July 26, 2002;10.1152/ajplung.00231.2001
Received 18 June 2001; accepted in final form 22 July 2002.
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