1 Division of Critical Care, Pulmonary, Allergic, and Immunological Diseases, Department of Medicine, Jefferson Medical College, Thomas Jefferson University; 2 Department of Therapeutics, Institute of Cell Signaling, University Hospital of Nottingham, Nottingham, United Kingdom NG7 2UH; 3 Division of Pulmonary and Critical Care, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104; and 4 Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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ABSTRACT |
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Numerous in vitro and in vivo
studies have implicated the cytokines interleukin-1 (IL-1
) and
tumor necrosis factor-
(TNF-
) as mediators of airway inflammation
and therefore potentially important substances in the pathogenesis of
asthma. In this study, we examined the mechanisms by which IL-1
and
TNF-
affect inhibition of cell growth, G protein-coupled receptor
(GPCR) desensitization, and the recently reported adenylyl cyclase
sensitization in human airway smooth muscle (HASM) cultures. Our
findings demonstrate that adenylyl cyclase sensitization is independent
of cytokine-mediated cyclooxygenase type 2 (COX-2) and prostaglandin
E2 (PGE2) induction, whereas COX-2 induction
appears to be required for both growth inhibition and GPCR
desensitization. However, GPCR desensitization was highly dependent on
the presence of EGF during chronic treatment with cytokines, which
could be explained by a synergistic effect of EGF on cytokine-mediated
COX-2 and PGE2 induction. Interestingly, various agents
(including inhibitors of p42/p44 and p38 mitogen-activated protein
kinase signaling) were significantly more effective in inhibiting
cytokine-mediated PGE2 induction, GPCR desensitization, and
cell growth inhibition than in inhibiting COX-2 induction. These data
demonstrate disparity in the requirement and sufficiency of COX-2
induction in promoting different functional effects of IL-1
and
TNF-
in HASM.
interleukin-1; tumor necrosis factor-
; adenylyl cyclase; sensitization
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INTRODUCTION |
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IN BRONCHOALVEOLAR
LAVAGE FLUID harvested from asthmatic subjects, interleukin-1
(IL-1
) and tumor necrosis factor-
(TNF-
) levels are elevated
(8, 23). Several in vitro studies have demonstrated the
effects of these cytokines on airway cells (including resident stromal
cells and infiltrating blood cells) that are consistent with a role for
IL-1
and TNF-
as mediators of airway inflammation. These effects
include increased adhesion-molecule expression, cytokine and matrix
secretion, and cell activation and chemotaxis (reviewed in Refs.
1, 12, and 15).
In airway smooth muscle (ASM) cultures, chronic IL-1 treatment has
been shown to inhibit mitogen-induced cell growth (3) and
promote hyporesponsiveness of
2-adrenergic receptors
(
2-ARs) as defined by diminished inhibition of cell contraction
(19, 21, 24, 33) and decreased cAMP production (14,
19, 21, 24, 28, 33) in response to
-agonists. It has been
suggested that these effects are mediated through an induction of
cyclooxygenase type 2 (COX-2) expression and the associated increase in
prostaglandin E2 (PGE2) production (3,
21). COX catalyzes the conversion of arachidonic acid to
PGH2, which is subsequently converted to prostanoids
including PGE2, PGI2, and thromboxane
A2. Of the two principal COX isoforms, COX-1 appears to be
constitutively expressed in most cell types and is responsible for
prostanoid synthesis under physiological conditions, whereas COX-2 is
induced by various inflammatory stimuli (reviewed in Ref.
21). In numerous cell types including ASM, IL-1
treatment has been shown to increase COX-2 protein expression as well
as cause accumulation of PGE2 in cell-culture media in an
indomethacin-sensitive manner. Because treatment of ASM cultures with
PGE2 also results in inhibition of ASM growth and
2-AR
hyporesponsiveness (3, 21), these collective findings
strongly implicate PGE2 generated as a result of COX-2
induction as the mediator of these cytokine-induced effects.
However, we have recently reported that chronic treatment of human ASM
(HASM) cultures with IL-1 also results in sensitization of adenylyl
cyclase (AC) (5). Because AC has been suggested to
represent the rate-limiting component in cAMP production mediated by
the G protein-coupled receptor (GPCR)-Gs protein-AC
transmembrane signaling cascade (4), sensitization appears
to be a homeostatic mechanism that mitigates the deleterious effect of
cytokines on
2-AR responsiveness to preserve
-agonist-stimulated
cAMP production (5).
In this study, we compare the mechanisms by which IL-1 and TNF-
promote the induction of COX-2 and PGE2, inhibition of cell growth, GPCR desensitization, and AC sensitization in HASM cultures. Our findings demonstrate that AC sensitization is independent of
cytokine-mediated COX-2 and PGE2 induction, whereas
COX-2 induction appears to be required for both growth inhibition
and GPCR desensitization. However, GPCR desensitization was highly
dependent on the presence of mitogenic growth factors such as epidermal
growth factor (EGF) during chronic treatment with cytokines, which
could be explained by a synergistic effect of EGF on cytokine-mediated
COX-2 and PGE2 induction. Interestingly, various agents
[including inhibitors of p42/p44 and p38 mitogen-activated protein
kinase (MAPK) signaling] were significantly more effective in
inhibiting cytokine-mediated PGE2 induction, GPCR
desensitization, and cell growth inhibition than in inhibiting COX-2
induction. These findings demonstrate disparity in the requirement and
sufficiency of COX-2 induction in promoting different functional
effects of IL-1
and TNF-
in HASM.
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METHODS |
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Materials. The Biotrak 125I-PGE2 RIA kit, ECL reagents, and [methyl-3H]thymidine (1 µCi/ml) were purchased from Amersham (Arlington Heights, IL). Human anti-COX-2 antibody was purchased from Oxford Biomedical Research (Oxford, MI). 125I-adenosine 3',5'-cyclic phosphoric acid (2,200 Ci/mmol) and [125I]iodopindolol (PIN; 2,200 Ci/mmol) were purchased from NEN-Dupont (Boston, MA). U-0126 was a gift from Dupont Pharmaceuticals (Wilmington, DE). cAMP antibody was provided by Mario Ascoli (University of Iowa, Iowa City, IA). SB-203580 and SB-202474 were purchased from Calbiochem (San Diego, CA). All other reagents were purchased from Sigma (St. Louis, MO) or from previously identified sources (26, 30).
HASM cell culture. HASM cultures were established as described by Panettieri et al. (27) from human tracheae obtained from lung-transplant donors in accordance with procedures approved by the University of Pennsylvania Committee on Studies Involving Human Beings. Characterization of this cell line with regard to immunofluorescence of smooth muscle actin and agonist-induced changes in cytosolic calcium has been previously reported (27).
Cells of passages 2-6 were plated at a density of 104 cells/cm2 in either 24-well ([3H]thymidine and cAMP assays) or 6-well (COX-2 protein, PGE2, and MAPK assays) plates in fetal bovine serum (FBS)-supplemented medium as described previously (26, 30). Seven days later, cells were growth-arrested by washing them with phosphate-buffered saline (PBS) and refeeding them with Ham's F-12 medium supplemented with 5 µg/ml each of insulin and transferrin (IT medium) for 48 h.Assay of cAMP accumulation.
HASM cultures were growth-arrested for 48 h as
described and treated with vehicle or indicated inhibitors for 30 min
before being treated with either 20 U/ml IL-1, 10 ng/ml TNF-
, or
both in the presence or absence of 10 ng/ml EGF for 18 h. Cultures were then washed thoroughly with cold PBS and subsequently stimulated with 500 µl of PBS containing 300 µM ascorbic acid, 1 mM Ro-20-1724 (a phosphodiesterase inhibitor), and either vehicle (basal), 1 µM
isoproterenol (Iso), 10 nM or 1 µM PGE2, or 100 µM
forskolin (Fsk) for 10 min at 37°C. cAMP was isolated and quantified
by radioimmunoassay using an 125I-cAMP tracer and cAMP
antibody (31) as described previously (30).
Radioligand binding.
To assess possible changes in 2-AR density and distribution in HASM
cells mediated by chronic cytokine + EGF treatment, cells were
grown to confluence in 15-cm dishes, growth-arrested for 48 h,
then treated for 18 h with cytokine + EGF as described. Cells
were washed in cold PBS and harvested as described previously (30). Approximately 100 µg of resuspended cells were
incubated with 200 pM [125I]PIN in the presence or
absence of 1 µM alprenolol (for determination of whole cell
2-AR
density) or 100 nM CGP-12177 (cell-surface
2-ARs) at 14°C for
3.5 h, and reactions were terminated via filtration through
Whatman GF-C filters using a Brandel cell harvester as described
previously (30).
Assay of [3H]thymidine incorporation and cell proliferation. Confluent growth-arrested cells were pretreated with inhibitors or the matched vehicle and were stimulated with various agents as indicated. After 16 h of stimulation, cells in 24-well plates were labeled with 3.0 µCi [methyl-3H]thymidine (1 µCi/ml) and incubated at 37°C for 24 h. Cells were then washed with PBS, harvested with 0.05% trypsin in 0.53 mM EDTA, and lysed with 20% trichloroacetic acid. The precipitate was aspirated onto filter paper and counted in scintillation vials. Data points from individual [3H]thymidine experiments represent the mean values derived from six wells.
Analysis of COX-2 expression and PGE2 production. HASM cultures were grown in six-well plates and treated exactly as described for cAMP assays. After 18 h of treatment with cytokines, the plates were placed on ice and 250 µl of media were harvested from each well for subsequent analysis of PGE2 concentration. The rest of the media were then aspirated from the well, the well was washed once with cold PBS, and 200 µl of 1% sodium dodecyl sulfate (SDS) sample buffer (26) were applied directly to the well. Cell lysates were harvested by scraping and subsequently analyzed by immunoblotting for COX-2 expression using a 1:2,000 dilution of primary antibody. PGE2 from the harvested culture media was purified using C-18 columns as described previously (17) and subsequently quantified using the Biotrak RIA kit as per the manufacturer's instructions.
Analysis of p42/p44, p54 c-Jun amino-terminal kinase, and p38 MAPK phosphorylation. HASM cells were plated in six-well plates as described (see HASM cell culture) and stimulated with various agents for 0-18 h. At the indicated time points, cells were washed once with cold PBS and then lysed by addition of SDS sample buffer. Lysates were analyzed by immunoblotting using antibodies that specifically recognize the phosphorylated forms of p42/p44, p54 c-Jun amino-terminal kinase (JNK), and p38 MAPK, as described previously (26). To confirm uniformity of gel loading, blots were stained with 0.2% Ponceau S, or parallel blots were run and probed with antibodies that recognize both phosphorylated and nonphosphorylated forms of the respective MAPKs.
Data presentation and statistical analysis. Data points from individual assays represent the mean values from duplicate or triplicate measurements. Statistical analysis of data was performed using GraphPad Prism Software (San Diego, CA) software. Except where noted, data are presented as means ± SE. EGF- and cytokine-mediated effects on basal and agonist-stimulated cellular cAMP accumulation are reported in Fig. 2. Agonist-stimulated cellular cAMP production was calculated as the cAMP measured in agonist-treated cells minus the matched basal value. To minimize interexperimental variability in subsequent experiments that examined the effect of various inhibitory agents, group values were normalized to the appropriate matched control value (either vehicle-treated or vehicle + EGF-treated groups). For PGE2-stimulated group data, the data reflecting values for 10 nM PGE2 stimulation are presented. However, experimental effects on cells stimulated with 1 µM PGE2 were qualitatively similar (data not shown). Statistically significant differences among groups were assessed by either ANOVA with Fisher's protected least-significant difference post hoc analysis (StatView 4.5, Abacus Concepts, Berkeley, CA) or by t-test for paired samples with P < 0.05 sufficient to reject the null hypothesis.
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RESULTS |
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Effects of cytokine on cell growth, GPCR desensitization, and AC
sensitization.
Initial studies characterized the effects of chronic treatment with
IL-1, TNF-
, or both on cell growth and GPCR-AC signaling in HASM
cultures. Chronic IL-1
, TNF-
, or IL-1
+ TNF-
treatment had little or no effect on HASM [3H]thymidine
incorporation (data not shown and Ref. 26). Consistent with previous observations noting the inhibitory effects of cytokines on serum-stimulated DNA synthesis in HASM cultures (3),
EGF-stimulated DNA synthesis was significantly inhibited by IL-1
or
combined IL-1
+ TNF-
(but not TNF-
alone) treatment, and
these effects were reversed by prior treatment with 1 µM
indomethacin, which suggests a dependence on COX activity (Fig.
1). On the basis of the results of
previous studies (14, 28, 33) and our previous analyses of
mechanisms of GPCR desensitization in HASM (25, 30) which
would suggest that the cytokine-promoted induction of COX-2 and the
associated PGE2 production should cause a desensitization of both the
2-AR and PGE2 receptors in ASM, we examined
the responsiveness of these receptors (defined by agonist-stimulated
cAMP generation) after chronic cytokine treatment. In contrast with
previous studies (14, 28, 33), chronic treatment with
IL-1
did not decrease Iso- or PGE2-stimulated cAMP
production relative to that stimulated in the control (vehicle-treated)
group (Fig. 2, B and
C). Similarly, chronic treatment with TNF-
failed to
inhibit agonist-stimulated cAMP generation. Only combined treatment
with IL-1
and TNF-
resulted in a loss of Iso- and
PGE2-stimulated cAMP generation. However, concomitant
treatment with EGF rendered IL-1
effective in inhibiting Iso- and
PGE2-stimulated cAMP generation and also increased the
inhibitory effect of IL-1
+ TNF-
treatment. Cells cotreated
with EGF and either IL-1
or IL-1
+ TNF-
exhibited significantly lower agonist-stimulated cAMP values than those of the
matched vehicle-treated (with EGF) group (Fig. 2, B and C). In addition, cAMP values for the EGF + IL-1
and
EGF + IL-1
+ TNF-
groups were also significantly lower
than those of the vehicle-treated (without EGF) group for each agonist,
with the exception being that Iso-stimulated cAMP values in the
EGF + IL-1
group were reduced (but not significantly so)
relative to the vehicle-treated (with EGF) group (22.1 ± 2.1 vs.
18.5 ± 1.9 pmol/well, respectively). In contrast, EGF treatment
failed to confer any desensitizing effect of TNF-
treatment. Despite
the clear effects of the treatment conditions on DNA synthesis
(measured after 40 h of treatment; see Fig. 1), protein content
per well (measured after 18 h of treatment) did not vary
significantly among the treatment groups (data not shown), which
suggests that alterations in cell number or whole-cell protein content
did not contribute to the observed experimental effects on cAMP
production.
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Effect of COX inhibition on GPCR desensitization and AC
sensitization.
Prior treatment with indomethacin reversed the GPCR desensitization
exhibited under both EGF-free and EGF-containing conditions, which
again suggests a dependence on COX activity (Fig.
4, A-D). However,
under all treatment conditions, cytokine-promoted AC sensitization was
minimally affected by prior indomethacin (Fig. 4, E and
F) or dexamethasone (data not shown) treatment, which suggests a COX-independent mechanism. Similarly, the effects of cytokine on basal cAMP levels were not altered by indomethacin treatment (data not shown).
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Effects of EGF on cytokine-mediated COX-2 and PGE2
induction.
Given the potentiating effect of EGF on cytokine-mediated GPCR
desensitization, we next examined the effects of EGF on COX-2 and
PGE2 induction by cytokines. In the absence of EGF, COX-2 protein levels were undetectable in both vehicle- and TNF--treated cells. IL-1
-induced COX-2 expression and combined IL-1
+ TNF-
treatment typically increased COX-2 expression to a level more than twofold that elicited by IL-1
treatment alone (Fig.
5A). Treatment with EGF alone
had a small effect on COX-2 expression that was observable only upon
overexposure of autoradiographs (data not shown). However, EGF
significantly increased both IL-1
- and IL-1
+ TNF-
-induced COX-2 expression and permitted a small but observable
induction of COX-2 expression by TNF-
. A similar potentiation of
COX-2 synthesis was observed in HASM cells treated with a combination
of cytokine(s) and either platelet-derived growth factor or 10%
(final) FBS (data not shown).
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Cytokine- and EGF-stimulated activation of p42/p44, p38, and p54
JNK MAPK.
Because previous studies in various cell types [including HASM
(19, 20)] have implicated roles for both p42/p44 and p38 in both the induction of COX-2 expression and related functional effects elicited by cytokines, we examined the time-dependent activation of the MAPK pathways by IL-1, TNF-
, IL-1
+ TNF-
, EGF, and combined EGF + IL-1
treatments. Acute
activation (30 min) of p42/p44 tended to be comparable among cells
stimulated with IL-1
, TNF-
, IL-1
+ TNF-
, EGF, or
EGF + IL-1
(Fig. 6A). At 3 h and 6 h, activation by IL-1
or TNF-
was still
detectable, and activation by IL-1
, TNF-
, EGF, and EGF + IL-1
was significantly greater. By 12 h and 18 h, a weak
activation by IL-1
and TNF-
was still detectable; activation by
EGF and to a lesser extent EGF + IL-1
was still prominent. For
p38, 30 min of treatment with IL-1
or TNF-
promoted p38
phosphorylation, but phosphorylation induced by EGF was minimal (Fig.
6B) as described previously (26). Combined
IL-1
+ TNF-
treatment resulted in a slightly greater phosphorylation of p38 than either IL-1
or TNF-
alone, whereas the effect of EGF + IL-1
was comparable to that of IL-1
alone. At the 3 h and 6 h time points, p38 phosphorylation by
either IL-1
or TNF-
was minimal, but both IL-1
+ TNF-
as well as EGF + IL-1
continued to promote a strong
phosphorylation of p38. Analysis of later time points failed to reveal
any significant activation of p38 by any of the stimuli beyond basal
levels. For p54 JNK, stimulation with IL-1
, TNF-
, IL-1
+ TNF-
, and EGF + IL-1
all significantly increased p54 JNK
phosphorylation at the 30-min time point, with IL-1
+ TNF-
treatment promoting a slightly greater signal than either IL-1
or
TNF-
alone (Fig. 6C). No clear activation of p54 JNK was
observed at later (>3 h) time points.
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Effects of p42/p44 and p38 MAPK inhibition on cytokine-mediated
effects.
To assess the requirement for p42/p44 and p38 MAPK activity, we
examined the effects of inhibiting p42/p44 [using the
mitogen-activated protein kinase/extracellular signal-regulated kinase
MEK1/2 inhibitor U-0126 (13)] and p38 [using SB-203580
(9)] on cytokine-mediated effects in HASM. Prior
treatment with 10 µM U-0126 or 1 µM SB-203580 significantly
reversed the cytokine-mediated inhibition of Iso- and
PGE2-mediated cAMP production (Fig.
7A-D) as did pretreatment with
indomethacin (see Fig. 4) or dexamethasone (not shown). In reversing
the observed GPCR desensitization, 1 µM U-0126 and 100 nM SB-203580
were ~50% as effective as 10 µM U-0126 and 1 µM SB-203580, respectively (data not shown). Interestingly, pretreatment with 1 µM
SB-202474 [an analog of SB-203580 that lacks the ability to inhibit
p38 (22)] was also able to partially reverse
cytokine-mediated 2-AR- and PGE2-receptor
desensitization. Conversely, U-0126, SB-203580, and
SB-202474 failed to inhibit cytokine-promoted AC sensitization
(Fig. 7, E and F). In fact, pretreatment with
SB-203580 or SB-202474 alone caused a slight increase in Fsk-stimulated cAMP production. Cytokine-mediated effects on basal cAMP were largely unaffected by the inhibitors, although U-0126 and
SB-203580 each caused partial inhibition of the increase in basal cAMP
levels caused by IL-1
treatment (data not shown).
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DISCUSSION |
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The present study demonstrates that in HASM, cytokines regulate
receptor-mediated cAMP production via two independent mechanistically distinct processes (Fig. 10). Through
the induction of COX-2 and PGE2 synthesis that occurs via
pathways sensitive to p42/p44, p38, and nuclear factor (NF)-B
inhibition, cytokines (IL-1
or IL-1
+ TNF-
) indirectly
stimulate cAMP production via PGE2-receptor activation and
thereby modulate cell functions such as growth. PGE2-receptor activation ultimately leads to homologous
desensitization of PGE2 receptors and heterologous
desensitization of
2-ARs. The induction of COX-2-PGE2
synthesis and GPCR desensitization by cytokines is highly dependent on
costimulation with growth factors/mitogens such as EGF, which serves to
promote a more robust and sustained activation of p42/p44 and p38 MAPK
than that stimulated by cytokines alone. Through an undetermined
mechanism that is dissociated from COX-2-PGE2 induction and
is not dependent on p42/p44, p38, or NF-
B activation, cytokines
(IL-1
, TNF-
, or IL-1
+ TNF-
) promote the sensitization
of AC, which serves to augment cAMP production and minimize the impact
of GPCR desensitization on receptor-mediated cAMP production. AC
sensitization by cytokines is minimally affected by concomitant EGF
treatment, suggesting that EGF and cytokines may invoke similar
partially redundant mechanisms to sensitize AC.
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The general observations that cytokines induce COX-2 and
PGE2 synthesis, modulate ASM growth, and promote GPCR
desensitization have been demonstrated by numerous studies. Previous
studies using ASM cultures from various species have identified
IL-1-mediated inhibition of serum-stimulated growth (3)
as well as decreased
2-AR or PGE2-receptor-mediated cAMP
production (14, 19, 21, 24, 28, 33) and inhibition of cell
contraction and tension (19, 21, 24, 33). The role of
COX-2 in mediating the effects of IL-1
in inhibiting
serum-stimulated ASM growth and promoting
2-AR hyporesponsiveness is
supported by detailed analyses by the Knox laboratory of
IL-1
-mediated induction of COX-2 protein and activity, and
associated eicosanoid synthesis in both bovine (10) and
human (29) ASM cultures. Additional studies assessing IL-1
effects on ASM growth (3) and GPCR signaling and
function (21, 24, 28, 33) in ASM have also demonstrated
IL-1
-mediated COX-2 and PGE2 induction. In addition,
IL-1
-mediated growth inhibition, GPCR hyporesponsiveness, and COX-2
induction were all inhibited by glucocorticoid treatment (2, 24,
28, 34), and functional effects as well as PGE2
induction were similarly inhibited by indomethacin pretreatment
(3, 21, 28). Moreover, pharmacological inhibition of
p42/p44 and p38 MAPK pathways was shown to inhibit IL-1
-mediated
COX-2 and PGE2 synthesis and to reverse the associated loss
of Iso-mediated relaxation of ASM stiffness (19, 20). Because exogenous PGE2 could mimic the effects of IL-1
treatment, the collective evidence strongly implicates induced
COX-2-mediated PGE2 synthesis as the principal mechanism by
which IL-1
alters ASM growth and GPCR function.
The present study extends these observations and provides new insight
into mechanisms underlying cytokine-promoted COX-2 and PGE2
synthesis and the associated effects on GPCR responsiveness and cell
growth. An important new finding relates to the sensitivity of
cytokine-mediated effects to additional signaling input from growth
factors. This sensitivity was originally suggested by Belvisi et al.
(3), who reported that PGE2 release after
chronic IL-1 treatment was observed only in serum-fed (but not
serum-deprived) HASM cells. In a recent study, we also noted that
chronic IL-1
treatment resulted in a loss of Iso-stimulated cAMP
production in serum-fed but not serum-deprived HASM (5).
Our current findings demonstrate that IL-1
is not sufficient to
induce COX-2 and PGE2 such that GPCR-mediated cAMP
production is significantly inhibited, and requires additional
signaling input from either growth factors or TNF-
to promote such
an effect.
The insufficiency of IL-1 to attenuate GPCR-mediated cAMP production
is further explained by the sensitization or superactivation of AC that
occurs not only with treatment with IL-1
, but also with TNF-
.
Although a desensitization of
2-AR and PGE2 receptors caused by IL-1
treatment (in the presence or absence of EGF) is more
readily interpreted if one considers the accompanying sensitization of
AC (and assumes that increased intrinsic AC activity proportionally
increases Gs-coupled receptor-mediated cAMP generation), the functional consequence of this desensitization (reduced cAMP generation in response to Iso or PGE2) is effectively
reversed as a result of AC sensitization. Our finding of IL-1
- and
TNF-
-promoted AC sensitization is at odds with findings from
numerous other studies that have reported no effect of either chronic
IL-1
(14, 21, 24, 33) or TNF-
(11, 16)
treatment on AC responsiveness. Differences in species or culture
conditions may explain the disparity between our results and those of
others. Hakonarson et al. (14) examined rabbit ASM strips
in which different (muscarinic-receptor dependent and pertussis-toxin
sensitive) mechanisms appeared to be involved in mediating the effects
of cytokines on GPCR signaling. Emala et al. (11) observed
no effect of 72 h of TNF-
treatment on AC activity in canine
ASM, although a subsequent study from the same group noted a trend
toward increased AC activity (~46% mean increase in five
experiments) caused by 72 h of TNF-
treatment of HASM
(16). Studies from the Shore (33) laboratory
focus on examining regulation of cell stiffness in HASM cultures, and parallel analyses of GPCR-AC-stimulated cAMP production are performed under carefully matched conditions in which cells are harvested after
chronic cytokine treatment and replated shortly before performance of
cAMP assays. Given the sensitivity of cytokine-dependent effects to
growth factor (and other mitogenic) stimuli that is demonstrated in the
present study, such conditions may obscure detection of AC sensitization.
Interestingly, this sensitization of AC was only minimally inhibited by
pertussis toxin [which suggests a mechanism distinct from that
responsible for AC elicited by Gi-coupled receptors (4)] and was also minimally affected by indomethacin (the
present study), which demonstrates that this phenomenon is not
dependent on COX activity. As noted above, the observed sensitization
of AC by EGF treatment alone and the less-than-additive effect of concomitant EGF and cytokine treatment suggest that EGF and cytokines may share, to some degree, a common mechanism that promotes AC sensitization. However, inhibition of the mitogen-activated kinases p42/p44 (by U-0126) or p38 (by SB-203580) did not inhibit AC
sensitization caused by either EGF or cytokines, although it was highly
effective in inhibiting COX-2 induction, PGE2 production,
and GPCR desensitization. Importantly, these latter positive effects of
both U-0126 and SB-203580 occurred at inhibitor concentrations (10 µM
and 1 µM, respectively) that favor specificity toward the intended
targets (p42/44 and p38). Particularly problematic may be the
nonspecific effects of SB-203580, which can include direct inhibition
of COX-1 and COX-2 (7), thromboxane A2
synthase (7), or phosphoinositide-dependent protein kinase
1 (18), or enhancement of NF-B activity
(6) when used at concentrations >2 µM in intact cell experiments.
Last, our findings suggest an apparent insufficiency of COX-2 induction
to promote significant PGE2 production and the associated effects on ASM growth and GPCR desensitization. Analysis of the relative effects of U-0126, SB-203580, and SB-202474 on COX-2 versus
PGE2 induction supports this assertion. We noted a
significant level of COX-2 protein was still induced by treatment with
IL-1 + TNF-
, IL-1
+ EGF, or IL-1
+ TNF-
+ EGF when cells were pretreated with U-0126 or SB-203580,
yet the accompanying PGE2 production was essentially
eliminated by these agents. One interpretation of these results is that
a very high threshold level of COX-2 induction is required before
PGE2 production and the functional effects elicited by
cytokines can occur. However, this does not appear to be the case,
because IL-1
induces a similar level of COX-2 induction compared
with that induced by IL-1
+ EGF in the presence of U-0126 or
SB-203580 but causes a much greater induction of PGE2.
Moreover, SB-202474, the analog of SB-203580 that lacks the ability to
inhibit p38, causes no inhibition of cytokine-induced COX-2 yet
inhibits PGE2 production by ~75%. These findings
challenge the dogma that COX-2 is limiting and is the critical factor
in the production of PGE2 in ASM, and suggest that the
activity of another element in the pathway that promotes increased
PGE2 synthesis is upregulated and that this activity can be
inhibited by agents that target p42/p44 (U-0126) or some other molecule
(that is inhibited by U-0126, SB-203580, or SB-202474) to inhibit the
functional consequences of cytokines on ASM. Within this context, the
role for U-0126, SB-203580, and SB-202474 in directly inhibiting enzyme activity or the induction of enzymes, cofactors, or possible complex formation important to PGE2 synthesis remains to be
established and represents an interesting challenge for future studies.
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ACKNOWLEDGEMENTS |
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We acknowledge Kristin Brodbeck and Andrew Eszterhas for valued technical assistance.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-58506 (to R. B. Penn) and HL-64063 (to R. A. Panettieri) and National Institute of Allergy and Infectious Diseases Grant AI-24509 (to S. P. Peters).
R. M. Pascual is recipient of a Glaxo Wellcome Pulmonary Fellowship, a Merck Young Investigator Award, and a Parker B. Francis Fellowship. R. B. Penn is a recipient of the American Lung Association Career Investigator Award.
Address for reprint requests and other correspondence: R. B. Penn, Thomas Jefferson Univ., Kimmel Cancer Institute, Rm. 930 BLSB, 233 S. 10th St., Philadelphia, PA 19107 (E-mail: rpenn{at}lac.jci.tju.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.
Received 16 January 2001; accepted in final form 3 August 2001.
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