| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
B-Dependent Signaling Pathway That Is Only Partially Sensitive to Dexamethasone1
Pulmonary, Allergy, and Critical Care Division, Department of Medicine, University of Pennsylvania Medical Center, Philadelphia, PA 19104
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B. In some cell types, NF-B activation is necessary to
regulate cytokine-mediated cellular functions. However, compelling
evidence suggests that the steroid inhibition of NF-B is complex and
cell specific. Using EMSA, we show that stimulation with TNF-
or
IL-1ß induces NF-B DNA-binding activity in human airway smooth
muscle cells. TNF- and IL-1ß also increased luciferase activity in
airway smooth muscle cells transfected with a reporter plasmid
containing B enhancer elements. Cytokines activated NF-B by
rapidly degrading its cytosolic inhibitor IB, which was then
regenerated after 60 min. Cytokine-mediated IB reappearance was
completely blocked by the protein synthesis inhibitor cycloheximide.
Inhibition of cytokine-mediated IB proteolysis using the protease
inhibitors N-tosyl-L-phenylalanine
chloromethyl ketone and
N-acetyl-L-leucinyl-L-leucinyl-norleucinal
also inhibited cytokine-mediated early expression of ICAM-1. Although
dexamethasone partially inhibited IL-1ß- and TNF--induced
up-regulation of ICAM-1 at 4 h, dexamethasone had no effect on
cytokine-induced ICAM-1 expression at 18–24 h. In addition, neither
cytokine-induced degradation or resynthesis of IB nor NF-B
DNA-binding activity were affected by dexamethasone. In cells
transfected with the luciferase reporter, dexamethasone did not affect
TNF--induced NF-B-dependent transcription. Interestingly,
cytokine-mediated expression of cyclooxygenase-2 was completely
abrogated by dexamethasone at 6 h. Together, these data
demonstrate that cytokine-mediated NF-B activation and ICAM-1
expression involve activation of a steroid-insensitive
pathway. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B transcription factors regulate myriad cellular processes
that include cell proliferation, apoptosis, and differentiation
(1, 2). Rel/NF-B regulates transcription by direct
binding to decameric sequences (B motifs) located in the promoters
and enhancers of a variety of viral and cellular genes, particularly
those involved in inflammatory responses. In many cell types,
Rel/NF-B exists in the cytoplasm as an inactive form through
association with inhibitory proteins called inhibitors of NF-B
(IBs)3 (reviewed in
Refs. 3 and 4). In the unstimulated cell,
IB masks the NF-B nuclear localization signal. Treatment of cells
with mitogens, growth factors, or cytokines promotes rapid nuclear
translocation of Rel/NF-B by a mechanism in which IB kinases,
IKK-1 and IKK-2, phosphorylate conserved serine residues within the
amino terminus of the IB and IBß, which then targets IB
for ubiquitin-dependent degradation by the 26S proteasome (1, 2, 5). NF-B inactivation occurs either by replenishing pools of
IB by NF-B-dependent stimulation of de novo synthesis of
IB or by the regulated phosphorylation of IBß, whose
expression is NF-B-independent. Despite research that has markedly
improved our understanding of the mechanisms by which cytokines
activate NF-B, arguably the most important unanswered questions
still focus on identifying the upstream signaling events coupling
cytokine receptors to activation of NF-B.
Compelling evidence suggests that the role of NF-B in cytokine
signaling and the attendant cellular responses are highly cell- and
tissue-specific (1, 2, 6). In hemapoietic cells, TNF-
activates NF-B and AP-1 and induces apoptosis in an
NF-B-dependent manner (1). In many different cell
types, including lymphocytes, NF-B also inhibits cell death
(7, 8, 9). Glucocorticoids, which are important
antiinflammatory drugs in the treatment of asthma, markedly inhibit
TNF--induced NF-B activation in HeLa, Jurkat, and 2B4 cells
(10, 11, 12, 13, 14). One mechanism by which glucocorticoids inhibit
NF-B activation is by increasing transcription of IB gene
expression. IB is an inhibitory protein that binds and
inactivates NF-B. In some cell types, glucocorticoids are
ineffective in abrogating cytokine-induced NF-B activation
(15, 16). These studies suggest that the role of NF-B
in cytokine signaling is unique to the cell and tissue studied.
NF-B is implicated as a pivotal transcription factor mediating
chronic inflammatory responses in asthma, rheumatoid arthritis,
psoriasis, and inflammatory bowel disease (17). Activation
of NF-B stimulates recruitment and activation of T and B
lymphocytes, eosinophils, and macrophages, which amplify and perpetuate
the inflammatory lesion. Despite considerable interest in studying the
role of NF-B in regulating immunocyte function, few investigators
have studied the role of NF-B in regulating functional processes in
structural cells such as smooth muscle and fibroblasts. In a variety of
diseases, smooth muscle cells can play an important immunomodulatory
role. Although TNF- and IL-1ß mediate some effects, in part,
through activation of NF-B, other transcription factors likely play
critical roles in transducing cytokine effects especially in
nonhemapoietic cell types. In cultured vascular smooth muscle cells,
the role of NF-B activation in mediating growth remains
controversial. Studies suggest that NF-B may or may not modulate
thrombin- and platelet-derived growth factor-induced vascular smooth
muscle cell proliferation (1, 2). NF-B-dependent
pathways also regulate oxidant-induced increases in A1 adenosine
receptor expression and TNF--induced expression of the bradykinin
(B1) receptor in vascular smooth muscle cells. In human airway smooth
muscle (ASM) cells, the role as well as the activation of NF-B in
cytokine signaling has not been investigated. Only one study from our
laboratory showed that cross-linking CD40, a member of the TNF-
receptor family, activates NF-B (18). In addition, a
variety of proinflammatory genes activated by TNF- and IL-1ß in
human ASM cells contain NF-B binding sites in their promoter region
(reviewed in Ref. 17). NF-B appears to play a central
role in mediating cytokine-induced synthetic functions in human ASM
cells.
In this study, we demonstrate that cytokine-mediated ICAM-1 expression,
in contrast to cyclooxygenase (COX)-2 expression, was not suppressed by
dexamethasone. In addition, dexamethasone had no effect on NF-B
activation following stimulation with TNF- and IL-1ß. Our data
show that the inability of dexamethasone to affect synthesis of ICAM-1
may be due, in part, to the cytokine activation of a
steroid-insensitive NF-B pathway.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Dexamethasone was purchased from Sigma (St. Louis, MO). HAM/F12,
FCS, trypsin, and antibiotics (penicillin and streptomycin) were
obtained from Life Technologies (Grand Island, NY). IL-1ß and TNF-
were purchased from Boehringer Mannheim (Minneapolis, MN).
Smooth muscle cell culture and characterization
The culture of human ASM cells was performed as described
elsewhere (19, 20). The characterization of the cultured
cells as smooth muscle cells was confirmed by immunostaining with
anti-smooth muscle -actin as previously described
(19).
EMSA
Nuclear extracts were prepared as described previously
(21). Briefly, cells grown in serum-free medium were
preincubated with or without dexamethasone and stimulated with TNF-
for 1 h at 37°C. The cells were then washed with ice-cold PBS
before they were resuspended in ice-cold lysis buffer (10 mM Tris-HCl,
pH 8, 60 mM KCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM DTT, 1 mM PMSF,
and 2 µg/ml leupeptin and aprotinin) for 15 min. Nuclei were
separated from the cytoplasm by centrifugation at 4°C for 5 min,
washed, and resuspended in nuclear extraction buffer (20 mM Tris-HCl,
pH 8, 400 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and
25% glycerol) for 10 min on ice. The resulting nuclear proteins (5–10
µg) were incubated with 3 µg of poly(dI-dC) and 0.4 pmol of
double-stranded 32P-labeled oligonucleotide
containing the NF-B consensus binding site
(5'-AGTTGAGCGGACTTTCCCAGGC-3'; Santa Cruz Biotechnology, Santa Cruz,
CA) in a total volume of 20 µl in a buffer containing 10 mM Tris, pH
7.5, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, and 5% glycerol. After 30 min at
room temperature, samples were separated on a nonreducing 4%
polyacrylamide gel using Tris running buffer (1 M, pH 7.5) containing 3
M sodium acetate and 0.5 M EDTA. The gels were dried and
autoradiographed with intensifying screens at -70°C.
Western blot analysis
Western blotting for IB and COX-2 were performed as
described previously (21, 22). Briefly, ASM cells were
washed with cold PBS and resuspended in lysis buffer containing 10 mM
Tris-HCl, pH 7.4, 0.5% sodium deoxycholate, 1 mM EDTA, 0.5% Nonidet
P-40, 1 mM PMSF, 1 mM
Na3VO4, and 10 µg/ml
aprotinin and leupeptin. The cell lysate was kept on ice for 20
min and centrifugated at 12,000 rpm for 5 min. The cytoplasmic proteins
(30 µg) were separated by SDS-PAGE on a 12.5% gel for IB and
an 8% gel for COX-2 proteins. Proteins were transferred onto
nitrocellulose membrane (Schleicher and Schuell, Keene, NH). The
membrane was then blocked for 1 h with 3% BSA in TBS and
incubated for 2 h with either rabbit polyclonal IgG
anti-IB Ab (Santa Cruz Biotechnology) or mouse monoclonal IgG
anti-COX-2 Ab (Cayman Chemical, Ann Arbor, MI). The appropriate
secondary HRP-conjugated Ab (Boehringer Mannheim, Minneapolis, MN) was
added and complexes detected by enhanced chemiluminescence (Amersham,
Arlington Heights, IL) and autoradiographed.
Flow cytometry analysis
Flow cytometry was performed as described previously with slight modifications (23). Briefly, adherent cells were washed with PBS, detached by trypsinization (2 min, 37°C) and then washed with Ham’s-F12 (10% FCS) media, centrifuged, and transferred to microfuge tubes (1.5 ml). Following incubation with the FITC-conjugated mouse anti-ICAM-1 Ab (10 µg/ml, R&D Systems, Minneapolis, MN) for 1 h at 4°C, the cells were centrifuged and resuspended in cold PBS in microfuge tubes. Samples were then analyzed using an EPICS XL flow cytometer (Coulter, Hialeah, FL). ICAM-1 expression was expressed as the increase in mean fluorescence intensity over background.
Transfection of human ASM cells
Transfections of human ASM cells were performed as described
previously (24). Briefly, ASM cells were transfected with
2 µg of pNF-B-Luc designed for monitoring the NF-B signal
transduction pathway (Clontech, Palo Alto, CA) and 2 µg of
pSV-ß-galactosidase control vector to normalize transfection
efficiencies (Promega, Madison, WI). Eighteen hours after transfection,
the cells were rendered quiescent in medium containing 0.2% FBS for
16 h and exposed to TNF- or IL-1ß for 4 h. Cells were
then harvested and luciferase and ß-galactosidase activities were
assessed as directed by Promega.
Statistical analysis
All data are expressed as means ± SEM. Comparisons between treatment were made using Student’s t test for paired values. The mean values were considered significantly different when the probability of the event was below 5%.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To examine critical signaling events that modulate
cytokine-mediated ICAM-1 expression, we examined whether
glucocorticoids inhibit cytokine-induced ICAM-1 expression in human ASM
cells. Previously, we showed that TNF- and IL-1ß induce expression
of ICAM-1 on human ASM (23, 25). TNF- induced a
7.1 ± 1-fold increase in ICAM-1 expression after 4 h that
was reduced to 3.7 ± 0.1 and 3.6 ± 0.2 in the presence of 1
or 10 µM dexamethasone, respectively (Fig. 1
A). IL-1ß induced a
5.3 ± 0.6-fold increase in ICAM-1 expression that was reduced to
3.14 ± 0.2 and 2.9 ± 0.2 in the presence of 1 or 10 µM
dexamethasone, respectively (Fig. 1A). In contrast, when
cells were exposed to cytokines for 24 h, dexamethasone failed to
suppress the expression of ICAM-1 (Fig. 1B). Dexamethasone
did not affect and even potentiated TNF--stimulated ICAM-1
expression after 24 h. The net increases in mean fluorescence over
background were 17.1 ± 0.6 (n = 4,
p < 0.01), 17.3 ± 1.3 (n = 4,
NS), and 18.7 ± 0.7 (n = 4, p <
0.05) in cells treated with TNF- alone and in the presence of
dexamethasone 0.01 µM and 1 µM, respectively. In contrast,
IL-1ß-mediated ICAM-1 expression after 24 h was partially
inhibited by dexamethasone with net increases in mean fluorescence over
background being 6.5 ± 0.6 (n = 4,
p < 0.01 vs basal), 6 ± 0.7 (n =
4, NS), and 5.4 ± 0.58 (n = 4, p
< 0.05) in cells treated with IL-1ß alone and in the presence of
dexamethasone 0.01 µM and 1 µM, respectively (Fig. 1B).
Taken together, these data clearly suggest that cytokine-mediated
ICAM-1 induction at early time points is partially sensitive to
glucocorticoids, while at later time points glucocorticoids have little
effect.
|
Previous studies in human ASM cells have shown that the rapid
COX-2 expression induced by IL-1ß is suppressed by dexamethasone
(22, 26). To ascertain whether concentrations of
dexamethasone used in our experimental conditions were effective, the
inducible expression of COX-2 proteins by IL-1ß was examined after
pretreating cells with 1 µM dexamethasone. After 6 h of
incubation with IL-1ß, there was a dramatic increase in the
expression of COX-2 by human ASM cells (Fig. 2). In contrast, TNF- had no effect on
COX-2 expression (data not shown). This effect is completely abrogated
by pretreatment of the cells with dexamethasone. Reprobing the same
blot with anti-COX-1 Ab revealed that COX-1 protein remained
unchanged in the three different conditions (Fig. 2). These data
suggest that 1 µM dexamethasone completely inhibits COX-2 expression
but has no effect on the induction of ICAM-1 by cytokines.
|
and IL-1ß induce NF-B activation in human ASM
To date, no studies have described the activation of transcription
factors by cytokines in human ASM cells. To study the effect of
cytokines on NF-B activation, ASM cells were transfected with a
construct consisting of B enhancer elements and a luciferase
reporter. Transfected cells were stimulated with cytokines and
luciferase activity in cell lysates was measured. Treatment of
transfected cells with TNF- or IL-1ß for 4 h induced a
3.4 ± 0.5- and 7.6 ± 1.6-fold increase in luciferase
activity, respectively, as compared with unstimulated cells (Fig. 3C). In contrast, neither
bradykinin nor thrombin, agonists that activate G-protein-coupled
receptors and mobilize calcium (20, 27), had any effect on
luciferase activity (Fig. 3C).
|
B activation is known to be a direct result of IB
degradation, its cytosolic inhibitor (3). Western blot
analysis of IB protein in cytoplasmic extracts of treated cells
demonstrated that TNF- (Fig. 3
A) or IL-1ß (Fig. 3B) induced IB degradation in a time-dependent manner,
which was almost complete by 15 min, with reappearance of IB
after 60 min. The resynthesis of IB was shown to be dependent
upon protein synthesis, because pretreatment of cells with
cycloheximide completely prevented the reappearance of IB after
60 min (Fig. 3B), without affecting IB degradation
(data not shown). These data suggest a potential role of NF-B in
cytokine-induced cellular responses in ASM cells.
Dexamethasone does not affect TNF--mediated IB
degradation, NF-B translocation, or NF-B-mediated transcription
In some cell types, dexamethasone inhibits cytokine-induced
NF-B activation. Therefore, we examined the ability of dexamethasone
to inhibit cytokine-mediated NF-B activation and IB
degradation. Pretreatment of ASM cells with dexamethasone had no effect
on TNF-- or IL-1ß-mediated IB degradation (Fig. 4A) or IB resynthesis
(Fig. 4B).
|
-mediated NF-B activation (Fig. 5A). In transfected cells with
an NF-B-luciferase reporter construct, we found that TNF- induced
a 4-fold increase in luciferase activity (Fig. 5B).
Pretreatment of the cells with dexamethasone had no effect on this
response (Fig. 5B). Taken together, these results
demonstrate that NF-B activation by cytokines in human ASM cells is
unaffected by concentrations of dexamethasone known to suppress NF-B
in other cell type (28).
|
B is necessary for cytokine-stimulated ICAM-1 but not COX-2
protein expression
Because functional NF-B regulatory elements are present within
the promoter region of the ICAM-1 gene (29, 30), we
hypothesized that NF-B activation is necessary for cytokine-mediated
effects on ICAM-1 expression in human ASM cells. We showed that
IB degradation is an obligatory step in the activation of
NF-B. To block NF-B activation, cells were pretreated with
protease inhibitors
N-tosyl-L-phenylalanine chloromethyl
ketone (TPCK) or
N-acetyl-L-leucinyl-L-leucinyl-norleucinal
(ALLN), which have been shown to prevent IB degradation and
NF-B translocation into the nucleus (reviewed in Ref.
4). The effect on ICAM-1 expression was examined.
Pretreatment of cells with TPCK (25 µM) or ALLN (50 µM) for 1
h abrogated both cytokine-mediated IB degradation (Fig. 6, A and B) and to
a similar extent the expression of ICAM-1. TPCK was found to be more
potent than ALLN in preventing IB degradation (Fig. 7). In contrast, we observed that
induction of COX-2 expression by IL-1ß was not affected by the same
concentration of TPCK (data not shown).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, that do not by themselves
induce ASM contraction can prime ASM cells to become hyperresponsive to
directly acting bronchoconstrictors (reviewed in Ref. 31).
In addition, these same cytokines can up-regulate smooth muscle cell
expression of the cell adhesion molecules ICAM-1 and VCAM-1, which can
support adhesion of activated leukocytes (23, 25). In this
study, we have characterized a potentially important signal
transduction pathway in ASM cells, which involves a
glucocorticoid-insensitive activation of NF-B by TNF- and
IL-1ß. Activation of NF-B is necessary for expression of ICAM-1,
but not for the expression of COX-2. In addition, dexamethasone
completely inhibited cytokine-induced COX-2 expression but not that of
ICAM-1. These data provide further evidence that IL-1ß and TNF-
induce activation of multiple signaling pathways for the induction of
gene expression.
Our initial studies focused on the ability of glucocorticoids to
inhibit TNF--induced ICAM-1 expression in ASM cells. We found that
at early time points glucocorticoids partially inhibited expression.
The mechanisms by which steroids affect early ICAM-1 expression induced
by cytokines are not delineated in the present study; however, several
hypotheses could be considered. In addition to NF-B binding sites,
the promoter region of the ICAM-1 gene contains various putative
transcriptional regulatory elements such as AP-1 and AP-1/Ets, AP-3,
and CCAAT/enhancer-binding protein (C/EBP) (29, 30).
The use of reporter plasmids and their 5' deletion derivatives
demonstrated that NF-B-dependent gene transcription is a rather
complex phenomenon and might involve a synergistic action of NF-B
with other transcription factors (32, 33, 34). Therefore, it
is possible that dexamethasone could suppress ICAM-1 expression at an
early time point by interfering with other transcription factors such
as C/EBP and AP-1 that have been shown to be involved in the
transcriptional regulation of the ICAM-1 gene. Support for this
hypothesis comes from investigators showing that dexamethasone, while
having a modest effect on NF-B activation, completely suppressed the
activation of AP-1 in response to IL-1ß (33). More
experiments are needed to determine the precise role of transcription
factors involved in ICAM-1 expression by cytokines to characterize the
effects of dexamethasone inhibition of TNF--induced ICAM-1
expression. Interestingly, the modest inhibitory effect of
dexamethasone on ICAM-1 expression was not sustained over 24 h.
Similar findings have been described in human hepatocytes
(35), human fibroblasts (36), and in vascular
endothelium (37, 38), where cytokine-induced ICAM-1
expression was either unchanged or only partially decreased by
steroids. In contrast, we and others have shown that dexamethasone
dramatically inhibits cytokine-induced COX-2 expression in ASM cells
(22, 26, 39). More recently, two studies performed in
human ASM cells reported that the expression and release of chemokines
IL-8 or RANTES in response to TNF- were partially inhibited by
dexamethasone (40, 41). This suggests that both
steroid-sensitive and -insensitive pathways are activated by cytokines
and modulate gene expression in ASM cells.
It is believed that many of the antiinflammatory effects of
glucocorticoids are due to their interference with transcription
factors, such as NF-B or AP-1 (reviewed in Ref. 17).
Therefore, we sought to determine the mechanisms underlying this
steroid-insensitive signaling pathway by studying the effects of
glucocorticoids on IL-1ß- and TNF--induced NF-B activation. We
found that neither NF-B DNA binding nor NF-B-mediated luciferase
activity was inhibited by dexamethasone pretreatment. This lack of
inhibition was seen even when the cells were preincubated with
dexamethasone for as long as 24 h. Further studies demonstrated
that both the time course of IB degradation and resynthesis were
unaffected by dexamethasone. The functional significance of IB
degradation in mediating cytokine-induced NF-B activation in human
ASM cells was examined by using the inhibitors TPCK and ALLN, which are
known to suppress NF-B activation by blocking IB degradation
(reviewed in Ref. 4). We found that pretreatment with TPCK
and ALLN inhibited cytokine-mediated IB degradation and abrogated
ICAM-1 expression in human ASM cells, without inducing changes in
cellular viability. This result is not entirely surprising because
NF-B regulatory elements are present within the promoter region of
the ICAM-1 gene, and studies have shown that NF-B activation is
critical for the regulation of ICAM-1 gene transcription (29, 30, 32, 36). Notably, however, TPCK and ALLN had no effect on
cytokine-induced COX-2 expression.
We found that not only is IL-1ß approximately two times more potent
than TNF- in activating NF-B reporter activity, but also that
IL-1ß, but not TNF-, stimulated COX-2 protein in human ASM cells.
Similarly, we have demonstrated that bradykinin, a calcium mobilizing
agent (20, 27), which can induce COX-2 expression in human
ASM cells (22), does not activate NF-B in human ASM
cells. In other cell types, IL-1ß induces COX-2 gene expression that
was associated with DNA-binding activity of AP-1, NF-B, and IFN-
activation site in keratinocytes (33). In mouse
osteoblastic cells, COX-2 induction involved NF-B activation but was
found to be 
80% regulated by the transcription factor NF-IL-6
(34). These data suggest that NF-B activation is
neither necessary nor sufficient to induce COX-2 expression in ASM
cells. In contrast, NF-B activation appears to be necessary for both
TNF-- and IL-1ß-induced expression of ICAM-1. Together, our
results therefore demonstrate that the signal transduction pathways
mediating cytokine-induced ICAM-1 expression in human ASM cells are
different from those used to stimulate COX-2 expression, and this may
explain their differential sensitivity to steroids.
Some reports describe that glucocorticoids act by direct
protein-protein interaction between the steroid receptor and NF-B
(13) or by the induction of IB protein (14, 28, 42). In contrast, others have demonstrated that steroids
modulate gene transcription through direct binding to glucocorticoid
responsive elements that regulate either the activation or repression
of gene transcription. Because the ICAM-1 gene lacks glucocorticoid
responsive elements in the promoter region (29, 30), it is
likely that steroids affect ICAM-1 through cross-coupling mechanisms
with NF-B or through the induction of IB. However, our data
demonstrate that glucocorticoids have no effect on either NF-B
DNA-binding activity or on IB synthesis, nor do they inhibit
cytokine-induced ICAM-1 expression. In contrast, COX-2 expression is
markedly diminished by glucocorticoids, which may represent the effects
on other transcription factors, such as AP-1, IFN- activation site,
or NF-IL-6, because the COX-2 promoter does not have a glucocorticoid
responsive element site (33). Similarly, several studies
have demonstrated that TNF- induces ICAM-1 transcription through
C/EBP and NF-B elements within the ICAM-1 promoter in endothelial
cells (32). TNF--mediated optimal activation of VCAM-1
promoter requires both Sp1 and NF-B (43). These data
suggest that at early time points, cytokines stimulate the differential
activation of transcription factors that may lead to different patterns
of gene transcription, such as ICAM-1 and COX-2 genes, and that may
express different sensitivity to the suppressive action of steroids.
Recently, investigators described that posttranscriptional mechanisms
involving a repression of COX-2 mRNA played an important role in
inhibiting IL-1ß-mediated COX-2 expression (44). These
findings suggest potential mechanisms, which are unrelated to the
modulation of gene transcription, can account for the effect of
dexamethasone on IL-1ß-induced COX-2 expression in human ASM cells.
However, additional experiments are needed to identify the precise
signaling events that mediate these effects.
After prolonged exposure of cells with cytokines, steroid effects on
ICAM-1 expression are more complex. Both TNF- and IL-1ß have been
shown to release a variety of proinflammatory mediators, such as
RANTES, IL-6, and IL-8, which are not completely blocked by
dexamethasone (40, 41). In our laboratory, we found that
TNF--induced RANTES expression was completely abrogated by
dexamethasone (our unpublished observations). Therefore, it is
plausible that the secreted proinflammatory agents could reduce steroid
action either by potentiating cytokine effects, as observed in this
study for TNF--induced ICAM-1 expression, or by impairing steroid
responsiveness. One recent study described that the release of NO in
mouse fibroblasts altered steroid responsiveness by decreasing both
steroid receptor affinity and number (45). Synthesis of NO
by human ASM cells has not been described, although a recent report
describes the ability of LPS to increase NO synthase mRNA in rat ASM
cells (46). Because glucocorticoids did not suppress
cytokine-induced ICAM-1 expression, it seems unlikely that steroid
modulation of the NO pathway plays a role in cytokine-induced ICAM-1
expression.
Modification of ASM function by cytokines has been regarded as a
potential mechanism underlying bronchial hyperresponsiveness in asthma
(reviewed in Ref. 31). Proinflammatory cytokines
up-regulate ASM expression of ICAM-1 and VCAM-1, which promotes
lymphocyte adhesion and induces smooth muscle cell DNA synthesis
(23). Increased levels of ICAM-1 have been detected in
asthmatic patients after allergen exposure (47, 48) or
during asthma attacks (49). mAbs against ICAM-1 are able
to inhibit both inflammatory cell infiltrate and acquired bronchial
hyperresponsiveness (50). In addition, a recent study
demonstrates the activation of NF-B DNA-binding activity in
bronchial biopsies obtained from patients with asthma
(51). Based on our data and those of others (40, 41), the inability of steroids to inhibit cytokine-induced
synthetic responses in ASM cells, involving the NF-B pathway, may
have important consequences for the local inflammatory response in
airways. Our data support the notion that the antiinflammatory effects
of corticosteroids in asthma may not be due to modulation of
cytokine-induced NF-B activation or ICAM-1 expression in human ASM
cells. Collectively, these studies open a new and important area of
investigation, delineating cytokine-mediated signal transduction
pathways that are insensitive to steroids.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Yassine Amrani, Pulmonary, Allergy, and Critical Care Division, Room 808 East Gates Building, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104-4283. E-mail address:
3 Abbreviations used in this paper: IB, NF-B inhibitor; ASM, airway smooth muscle; COX, cyclooxygenase; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; ALLN, N-acetyl-L-leucinyl-L-leucinyl-norleucinal.
Received for publication April 6, 1999. Accepted for publication May 28, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B transcription factors during lymphocyte activation. Curr. Opin. Immunol. 10:353.[Medline]
B. Immunol. Today 19:80.[Medline]
B and IB proteins: new discoveries and insights. Annu. Rev. Immunol. 14:649.[Medline]
B/IB family: intimate tales of association and dissociation. Genes Dev. 9:2723.B activation: the IB kinase revealed?. Cell 91:299.[Medline]
B in the immune system. Annu. Rev. Immunol. 12:141.[Medline]
B activation and regulates lymphocyte proliferation and survival. Immunity 7:703.[Medline]
B in preventing TNF--induced cell death. Science 274:782.B activation prevents cell death. Cell 87:565.[Medline]
B: what language?. Trends Biochem. Sci. 23:233.[Medline]
and interleukin 1 signalling: do MAPKK kinases connect it all?. Trends Pharmacol. Sci. 18:319.[Medline]
B kinase-ß. Nature 396:77.[Medline]
B in mediation of immunosuppression by glucocorticoids. Science 270:283.B activity through induction of IB synthesis. Science 270:286., and inhibition by glucocorticoids. Biochem. J. 328:707.
B (NF-B) and B-dependent transcription in epithelial cells. Eur. J. Biochem. 254:81.[Medline]
B—a pivotal transcription factor in chronic inflammatory diseases. N. Engl. J. Med. 336:1066.-p55 receptor induces myocyte proliferation and modulates agonist-evoked calcium transients in cultured human tracheal smooth muscle cells. Am. J. Respir. Cell Mol. Biol. 15:55.[Abstract]
of calcium signals induced by bradykinin and carbachol in human tracheal smooth muscle cells. Br. J. Pharmacol. 114:4.[Medline]
activates NF-B in acid sphinogmyelinase-deficient mouse embryonic fibroblasts. J. Biol. Chem. 272:10904. and interferon- on the induction of cyclo-oxygenase-2 in cultured human airway smooth muscle cells. Br. J. Pharmacol. 121:579.[Medline]
-induced ICAM-1 and VCAM-1 expression and T lymphocyte adhesion. J. Immunol. 154:2358.[Abstract]
effects on agonist-mediated calcium homeostasis in human airway smooth muscle cells. Am. J. Physiol. (Lung Cell. Mol. Physiol.) 273/17:L1020.B and inhibiting NF-B. Am. J. Physiol. 273:G1290. activate intercellular adhesion molecule-1 (ICAM-1) gene transcription through distinct cis-regulatory elements within the ICAM-1 promoter. J. Biol. Chem. 270:18966.B and nuclear factor-interleukin-6 in the tumor necrosis factor -dependent induction of cyclooxygenase-2 in MC3T3–E1 cells. J. Biol. Chem. 270:31315.B by activated glucocorticoid receptors. Mol. Cell. Biol. 15:943.[Abstract]
B, in asthma. Am. J. Respir. Crit. Care Med. 158:1585.This article has been cited by other articles:
|
|
 |
|
  M. Kaur, N. S. Holden, S. M. Wilson, M. B. Sukkar, K. F. Chung, P. J. Barnes, R. Newton, and M. A. Giembycz Effect of {beta}2-adrenoceptor agonists and other cAMP-elevating agents on inflammatory gene expression in human ASM cells: a role for protein kinase A Am J Physiol Lung Cell Mol Physiol, September 1, 2008; 295(3): L505 - L514. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Quante, Y. C. Ng, E. E. Ramsay, S. Henness, J. C. Allen, J. Parmentier, Q. Ge, and A. J. Ammit Corticosteroids Reduce IL-6 in ASM Cells via Up-Regulation of MKP-1 Am. J. Respir. Cell Mol. Biol., August 1, 2008; 39(2): 208 - 217. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Nehme and J. Edelman Dexamethasone Inhibits High Glucose-, TNF-{alpha}-, and IL-1{beta}-Induced Secretion of Inflammatory and Angiogenic Mediators from Retinal Microvascular Pericytes Invest. Ophthalmol. Vis. Sci., May 1, 2008; 49(5): 2030 - 2038. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Tliba, G. Damera, A. Banerjee, S. Gu, H. Baidouri, S. Keslacy, and Y. Amrani Cytokines Induce an Early Steroid Resistance in Airway Smooth Muscle Cells: Novel Role of Interferon Regulatory Factor-1 Am. J. Respir. Cell Mol. Biol., April 1, 2008; 38(4): 463 - 472. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Tliba, Y. Amrani, and R. A. Panettieri Jr Is Airway Smooth Muscle the "Missing Link" Modulating Airway Inflammation in Asthma? Chest, January 1, 2008; 133(1): 236 - 242. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Tliba and Y. Amrani Airway Smooth Muscle Cell as an Inflammatory Cell: Lessons Learned from Interferon Signaling Pathways Proceedings of the ATS, January 1, 2008; 5(1): 106 - 112. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Issa, S. Xie, N. Khorasani, M. Sukkar, I. M. Adcock, K.-Y. Lee, and K. F. Chung Corticosteroid Inhibition of Growth-Related Oncogene Protein-{alpha} via Mitogen-Activated Kinase Phosphatase-1 in Airway Smooth Muscle Cells J. Immunol., June 1, 2007; 178(11): 7366 - 7375. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. G. Tirumurugaan, J. A. Jude, B. N. Kang, R. A. Panettieri, T. F. Walseth, and M. S. Kannan TNF-{alpha} induced CD38 expression in human airway smooth muscle cells: role of MAP kinases and transcription factors NF-{kappa}B and AP-1 Am J Physiol Lung Cell Mol Physiol, June 1, 2007; 292(6): L1385 - L1395. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Keslacy, O. Tliba, H. Baidouri, and Y. Amrani Inhibition of Tumor Necrosis Factor-{alpha}-Inducible Inflammatory Genes by Interferon-{gamma} Is Associated with Altered Nuclear Factor-{kappa}B Transactivation and Enhanced Histone Deacetylase Activity Mol. Pharmacol., February 1, 2007; 71(2): 609 - 618. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Issa, S. Xie, K.-Y. Lee, R. D. Stanbridge, P. Bhavsar, M. B. Sukkar, and K. F. Chung GRO-{alpha} regulation in airway smooth muscle by IL-1beta and TNF-{alpha}: role of NF-{kappa}B and MAP kinases Am J Physiol Lung Cell Mol Physiol, July 1, 2006; 291(1): L66 - L74. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Lajoie-Kadoch, P. Joubert, S. Letuve, A. J. Halayko, J. G. Martin, A. Soussi-Gounni, and Q. Hamid TNF-{alpha} and IFN-{gamma} inversely modulate expression of the IL-17E receptor in airway smooth muscle cells Am J Physiol Lung Cell Mol Physiol, June 1, 2006; 290(6): L1238 - L1246. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B.-N. Kang, K. G. Tirumurugaan, D. A. Deshpande, Y. Amrani, R. A. Panettieri, T. F. Walseth, and M. S. Kannan Transcriptional regulation of CD38 expression by tumor necrosis factor-{alpha} in human airway smooth muscle cells: role of NF-{kappa}B and sensitivity to glucocorticoids FASEB J, May 1, 2006; 20(7): 1000 - 1002. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Tliba, J. A. Cidlowski, and Y. Amrani CD38 Expression Is Insensitive to Steroid Action in Cells Treated with Tumor Necrosis Factor-{alpha} and Interferon-{gamma} by a Mechanism Involving the Up-Regulation of the Glucocorticoid Receptor beta Isoform Mol. Pharmacol., February 1, 2006; 69(2): 588 - 596. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. H. Kim, D. Jain, O. Tliba, B. Yang, W. F. Jester Jr., R. A. Panettieri Jr., Y. Amrani, and E. Pure TGF-{beta} potentiates airway smooth muscle responsiveness to bradykinin Am J Physiol Lung Cell Mol Physiol, October 1, 2005; 289(4): L511 - L520. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. A. Deshpande, T. A. White, S. Dogan, T. F. Walseth, R. A. Panettieri, and M. S. Kannan CD38/cyclic ADP-ribose signaling: role in the regulation of calcium homeostasis in airway smooth muscle Am J Physiol Lung Cell Mol Physiol, May 1, 2005; 288(5): L773 - L788. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. L. Clarke, M. G. Belvisi, S. J. Smith, E. Hardaker, M. H. Yacoub, K. K. Meja, R. Newton, D. M. Slater, and M. A. Giembycz Prostanoid receptor expression by human airway smooth muscle cells and regulation of the secretion of granulocyte colony-stimulating factor Am J Physiol Lung Cell Mol Physiol, February 1, 2005; 288(2): L238 - L250. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Panettieri Jr. Effects of Corticosteroids on Structural Cells in Asthma and Chronic Obstructive Pulmonary Disease Proceedings of the ATS, November 1, 2004; 1(3): 231 - 234. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Tliba, R. A. Panettieri Jr., S. Tliba, T. F. Walseth, and Y. Amrani Tumor Necrosis Factor-{alpha} Differentially Regulates the Expression of Proinflammatory Genes in Human Airway Smooth Muscle Cells by Activation of Interferon-{beta}-Dependent CD38 Pathway Mol. Pharmacol., August 1, 2004; 66(2): 322 - 329. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. I. Darville, S. Terryn, and D. L. Eizirik An Octamer Motif Is Required for Activation of the Inducible Nitric Oxide Synthase Promoter in Pancreatic {beta}-Cells Endocrinology, March 1, 2004; 145(3): 1130 - 1136. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Peng, T. Matsuda, and S. J. Hirst Signaling Pathways Regulating Interleukin-13-stimulated Chemokine Release from Airway Smooth Muscle Am. J. Respir. Crit. Care Med., March 1, 2004; 169(5): 596 - 603. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Tliba, S. Tliba, C. Da Huang, R. K. Hoffman, P. DeLong, R. A. Panettieri Jr., and Y. Amrani Tumor Necrosis Factor {alpha} Modulates Airway Smooth Muscle Function via the Autocrine Action of Interferon {beta} J. Biol. Chem., December 12, 2003; 278(50): 50615 - 50623. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. S. Faffe, T. Whitehead, P. E. Moore, S. Baraldo, L. Flynt, K. Bourgeois, R. A. Panettieri, and S. A. Shore IL-13 and IL-4 promote TARC release in human airway smooth muscle cells: role of IL-4 receptor genotype Am J Physiol Lung Cell Mol Physiol, October 1, 2003; 285(4): L907 - L914. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. De Bosscher, W. Vanden Berghe, and G. Haegeman The Interplay between the Glucocorticoid Receptor and Nuclear Factor-{kappa}B or Activator Protein-1: Molecular Mechanisms for Gene Repression Endocr. Rev., August 1, 2003; 24(4): 488 - 522. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. A. Schroeder, J. M. Pearl, S. M. Schwartz, T. P. Shanley, P. B. Manning, and D. P. Nelson Combined Steroid Treatment for Congenital Heart Surgery Improves Oxygen Delivery and Reduces Postbypass Inflammatory Mediator Expression Circulation, June 10, 2003; 107(22): 2823 - 2828. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-D. Huang, O. Tliba, R. A. Panettieri Jr., and Y. Amrani Bradykinin Induces Interleukin-6 Production in Human Airway Smooth Muscle Cells: Modulation by Th2 Cytokines and Dexamethasone Am. J. Respir. Cell Mol. Biol., March 1, 2003; 28(3): 330 - 338. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Zhao and K. P. Karalis Regulation of Nuclear Factor-{kappa}B by Corticotropin-Releasing Hormone in Mouse Thymocytes Mol. Endocrinol., November 1, 2002; 16(11): 2561 - 2570. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Ammit, A. L. Lazaar, C. Irani, G. M. O'Neill, N. D. Gordon, Y. Amrani, R. B. Penn, and R. A. Panettieri Jr. Tumor Necrosis Factor-alpha -Induced Secretion of RANTES and Interleukin-6 from Human Airway Smooth Muscle Cells . Modulation by Glucocorticoids and beta -Agonists Am. J. Respir. Cell Mol. Biol., April 1, 2002; 26(4): 465 - 474. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Amrani, A. J. Ammit, and R. A. Panettieri Jr. Tumor Necrosis Factor Receptor (TNFR) 1, but Not TNFR2, Mediates Tumor Necrosis Factor-alpha -Induced Interleukin-6 and RANTES in Human Airway Smooth Muscle Cells: Role of p38 and p42/44 Mitogen-Activated Protein Kinases Mol. Pharmacol., October 1, 2001; 60(4): 646 - 655. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. PYPE, H. XU, M. SCHUERMANS, L. J. DUPONT, W. WUYTS, J. C. W. MAK, P. J. BARNES, M. G. DEMEDTS, and G. M. VERLEDEN Mechanisms of Interleukin 1{beta}-Induced Human Airway Smooth Muscle Hyporesponsiveness to Histamine . Involvement of p38 MAPK and NF-{kappa}B Am. J. Respir. Crit. Care Med., March 15, 2001; 163(4): 1010 - 1017. [Abstract] [Full Text]
|
|
|
|
|
|
A. J. Ammit, R. K. Hoffman, Y. Amrani, A. L. Lazaar, D. W. P. Hay, T. J. Torphy, R. B. Penn, and R. A. Panettieri Jr. Tumor Necrosis Factor-alpha -Induced Secretion of RANTES and Interleukin-6 from Human Airway Smooth-Muscle Cells . Modulation by Cyclic Adenosine Monophosphate Am. J. Respir. Cell Mol. Biol., December 1, 2000; 23(6): 794 - 802. [Abstract] [Full Text]
|
|
|
|
|
|
X. Liu and T. H. Piela-Smith Fibrin(ogen)-Induced Expression of ICAM-1 and Chemokines in Human Synovial Fibroblasts J. Immunol., November 1, 2000; 165(9): 5255 - 5261. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Amrani, A. L. Lazaar, R. Hoffman, K. Amin, S. Ousmer, and R. A. Panettieri Jr. Activation of p55 Tumor Necrosis Factor-alpha Receptor-1 Coupled to Tumor Necrosis Factor Receptor-Associated Factor 2 Stimulates Intercellular Adhesion Molecule-1 Expression by Modulating a Thapsigargin-Sensitive Pathway in Human Tracheal Smooth Muscle Cells Mol. Pharmacol., July 1, 2000; 58(1): 237 - 245. [Abstract] [Full Text]
|
|
|
|
|
|
P. E. Moore, T. L. Church, D. D. Chism, R. A. Panettieri Jr., and S. A. Shore IL-13 and IL-4 cause eotaxin release in human airway smooth muscle cells: a role for ERK Am J Physiol Lung Cell Mol Physiol, April 1, 2002; 282(4): L847 - L853. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
