Department of Pediatrics and Ben May Institute for Cancer Research, University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
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Ligation of the
asialoGM1 Pseudomonas aeruginosa pilin receptor has been
demonstrated to induce IL-8 expression in airway epithelial cells via
an NF-B-dependent pathway. We examined the signaling pathways
required for asialoGM1-mediated NF-
B activation in IB3 cells, a
human bronchial epithelial cell line derived from a cystic fibrosis
(CF) patient, and C-38 cells, the rescued cell line that expresses a
functional CF transmembrane regulator. Ligation of the
asialoGM1 receptor with specific antibody induced greater IL-8
expression in IB3 cells than C-38 cells, consistent with the greater
density of asialoGM1 receptors in CF phenotype cells. AsialoGM1-mediated activation of NF-
B, I
B kinase (IKK), and ERK
was also greater in IB3 cells. With the use of genetic inhibitors, we
found that IKK-
and NF-
B-inducing kinase are required for maximal
NF-
B transactivation and transcription from the IL-8 promoter.
Finally, although ERK activation was required for maximal asialoGM1-mediated IL-8 expression, inhibition of ERK signaling had no
effect on IKK or NF-
B activation, suggesting that ERK regulates IL-8
expression in an NF-
B-independent manner.
asialoGM1; extracellular signal-regulated kinase; IB kinase; mitogen-activated protein kinase; mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase kinase; nuclear
factor-
B-inducing kinase; tumor necrosis factor-
; cystic fibrosis
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INTRODUCTION |
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CHRONIC AIRWAY
INFLAMMATION is an important feature of cystic fibrosis (CF). The
concentrations of IL-8, TNF-, and other proinflammatory cytokines
have been noted to be increased in the sputum and bronchoalveolar
lavage of patients with CF (4, 8, 16, 23, 42).
Investigators have compared cytokine expression in airway epithelial
cells derived from CF individuals with those derived from normal
individuals. DiMango and colleagues (15) found a fourfold
increase in IL-8 secretion in response to Pseudomonas aeruginosa induction or ligation of the P. aeruginosa
pilin receptor, gangliotetraosylceramide (asialoGM1), in IB3
cells, an adenoassociated virus-transformed human bronchial
epithelial cell line derived from a CF patient (
F508/W1282X)
compared with its CF transmembrane regulator (CFTR)-corrected line C-38
cells. These authors later noted that IL-8 production was increased in
CF cells not only following stimulation but also in unstimulated cells
(14). Increased TNF-
-induced IL-8 expression in IB3
cells was confirmed by Venkatakrishnan and coworkers (46).
Similar results were recently found in two additional CF phenotypic
cell lines, pCEP-R, human tracheal epithelial cells stably transfected
with the regulatory domain of CFTR, and 16HBE-AS cells, human bronchial
epithelial cells transfected with antisense CFTR (24). In
both instances, CF cells demonstrated enhanced IL-8 protein expression
in response to bacterial induction, and, in the case of pCEP-R, basal
IL-8 production was also increased.
Tabary and coworkers (45) found that IL-8 expression is
also increased in CF primary bronchial gland epithelial cells both in
vivo and in vitro. On the other hand, Black and colleagues (3) found that IL-8 production in response to TNF- and
respiratory syncytial virus stimulation was not different in normal and
CF primary nasal epithelial cells. Massengale and co-workers
(32) found that CFT1 cells, a CF airway cell line,
secreted less IL-8 in response to IL-1
than an isogenic corrected
line (CFT1-LCFSN cells). Although differences in cell type and the
nature and duration of cell stimulation make comparisons difficult,
together these studies lend support to the view that mutant CFTR may
affect endogenous amounts of proinflammatory cytokine expression.
The mechanism by which cytokine expression may be upregulated in CF
cells has recently been studied. DiMango and colleagues (14) examined the contribution of NF-B to IL-8
expression in control (1HAEo
and C-38) and CF
(CFTEo
and IB3) cell lines. Relative to corrected or
control cells, the DNA binding of NF-
B was increased in both
unstimulated and stimulated CF cell lines. Venkatakrishnan and
colleagues (46) attributed increased NF-
B activation in
IB3 cells to increased basal levels of I
B
. Tabary and coworkers
(45) noted increased NF-
B activation and I
B
phosphorylation in CF airway gland cells. Schwiebert and colleagues
(43) demonstrated in airway epithelial cells that CFTR
activates the RANTES (for regulated upon activation, normal T cell
expressed, and secreted) promoter via an NF-
B-mediated pathway.
Finally, Weber and colleagues (48) recently demonstrated increased basal NF-
B transactivation in pCEP-R and 16HBE-AS CF phenotype cells. In addition, they found that expression of
F508 CFTR in Chinese hamster ovary cells increases NF-
B transcriptional activity, consistent with the notion that NF-
B plays a significant role in the dysregulated inflammatory response in CF.
Few studies have examined the upstream signaling intermediates
regulating NF-B activation in response to P. aeruginosa.
With the use of a series of chemical inhibitors, Li and colleagues (28) concluded that P. aeruginosa
lipopolysaccharide induces NCI-H292 pulmonary epithelial cell MUC2
expression via a Src/Ras/ERK/90-kDa ribosomal S6 kinase (RSK)/NF-
B
pathway. Denning and coworkers (10) showed that chemical
inhibition of two MAP kinase family members, ERK and p38, each
attenuated IL-8 expression in P. aeruginosa pyocyanin-treated A549 pulmonary epithelial cells. Similarly, Ratner
and colleagues (40), using a series of chemical
inhibitors, found that activation of NF-
B, ERK, and p38 was each
required for maximal IL-8 expression in 1HAEo
human
airway epithelial cells incubated with P. aeruginosa or antibody to asialoGM1. As far as we aware, there is no
information examining P. aeruginosa or anti-asialoGM1
antibody-induced signaling in CF phenotype cells.
In the present study, we hypothesized that IB kinase (IKK),
NF-
B-activating kinase (NIK), MAP kinase/ERK kinase kinase-1 (MEKK1), and ERK, each of which have been implicated in NF-
B signaling, would be required for P. aeruginosa-induced
NF-
B activation and IL-8 expression in IB3 CF phenotype cells. We
found that ligation of the asialoGM1 receptor with specific antibody
induced greater IL-8 expression in IB3 cells than C-38 cells,
consistent with the greater density of asialoGM1 receptors in CF
phenotype cells (5, 7, 20). AsialoGM1-mediated activation
of NF-
B, IKK, and ERK was also greater in IB3 cells. IKK-
and
NIK, but not MEKK1, were required for maximal NF-
B transactivation
and transcription from the IL-8 promoter. Finally, although ERK was
required for maximal IL-8 expression, inhibition of ERK signaling had
no effect on IKK-
or NF-
B activation, suggesting that ERK
regulates IL-8 expression in an NF-
B-independent manner.
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MATERIALS AND METHODS |
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Materials.
Anti-asialoGM1 antibody was purchased from Wako Chemical (Richmond,
VA). [-32P]ATP and an enhanced chemiluminescence kit
were obtained from DuPont/NEN Research Products (Wilmington, DE). Human
TNF-
was obtained from R&D Systems (Minneapolis, MN). Antibodies
against IKK-
and the NF-
B family transcription factors p65, p50,
c-Rel, and RelB were obtained from Santa Cruz Biotechnology (Santa
Cruz, CA). Myelin basic protein (MBP) was purchased from Sigma Chemical (St. Louis, MO). Activating transcription factor (ATF)-2 was obtained from Cell Signaling Technology (Beverly, MA). An antihemagglutinin (anti-HA) monoclonal antibody (HA.11) was purchased from Babco (Berkeley, CA). RNA was isolated using a RNAqueous-4PCR kit (Ambion, Austin, TX). RT-PCR reagents were obtained from Clontech (Palo Alto,
CA). An NF-
B reporter plasmid NF-
B-TATA-luc was purchased from
Stratagene (La Jolla, CA). TransIT-LT was obtained from Mirus (Madison,
WI). Optimem transfection medium was obtained from Invitrogen (Gaithersburg, MD). Luciferase assay buffer, U0126, and an
oligonucleotide probe encoding the consensus sequences of NF-
B were
purchased from Promega (Madison, WI). IL-8 ELISA reagents were obtained from Endogen (Woburn, MA).
Cell culture.
IB3-1 cells, an adeno-12-SV40-immortalized human bronchial epithelial
cell line derived from a CF patient (F508/W1282X), and C38 cells,
the rescued cell line that expresses an episomal copy of a truncated
but functional CFTR, were obtained from P. Zeitlin (Johns Hopkins
University, Baltimore, MD) (14, 51). Cells were grown in
LHC-8 (Biofluids, Rockville, MD) supplemented with 5% FBS.
Plasmid vectors.
The 162/+44 fragment of the full-length human IL-8 promoter,
subcloned into luciferase (
162/+44 hIL-8/Luc) (18), was
provided by A. Brasier (University of Texas, Galveston, TX). The
reporter activities of this fragment have been shown to be identical to the full-length promoter in response to respiratory syncytial virus
infection (18). Construction of cDNAs encoding dominant negative IKK-
(in which Ser-177 and -181 were replaced by alanines), NIK-KM (KK429/AA430), MEKK1-KM (K432M), and HA-tagged IKK-
has been
described elsewhere (34). A plasmid encoding dominant
negative (pCMV-MEK-2A) forms of MEK1, in which serine-218 and -222 phosphorylation sites were modified to alanine, was provided by D. Templeton (Case Western Reserve University) (50). A
bacterial expression vector encoding recombinant GST-I
B
was
provided by M. Karin (University of California, San Diego, CA)
(12). A cDNA encoding
-galactosidase was provided by M. Rosner (University of Chicago).
IL-8 protein abundance.
Confluent C38 and IB3 cells were serum starved for 24 h and
treated with either TNF- (10 ng/ml) or anti-asialoGM1 antibody (1:50) overnight. Growth media was collected and IL-8 protein was
measured by ELISA.
Immunoblotting. Cell lysates were resolved on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose by semidry transfer. After incubation with antibody, signals were amplified and visualized by enhanced chemiluminescence.
Reporter assays.
Cells were transfected with cDNAs encoding the NF-B reporter or IL-8
promoter using TransIT-LT, a polyamine transfection reagent. Cells were
grown to 50-80% confluence, washed in Optimem, and incubated with
a solution of plasmid DNA (~1.0 µg total DNA per 35-mm dish),
TransIT-LT (2 µl/dish), and Optimem. To normalize for transfection
efficiency, cells were cotransfected with a cDNA encoding
-galactosidase. After 3.5 h, the transfection solution was
replaced with 5% FBS/LHC-8. Twenty-four hours after transfection, cells were serum starved for 8 h and treated with either TNF-
(10 ng/ml) or anti-asialoGM1 (1:50). Sixteen hours after treatment, cells were harvested and analyzed for luciferase activity using a
luminometer, as described (39). Luciferase content was
assessed by measuring the light emitted during the initial 30 s of
the reaction, and the values were expressed in arbitrary light units. The background activity from cell extracts was typically <0.2 units,
compared with basal signals on the order of 102 units.
-Galactosidase activity was assessed by colorimetric assay using
o-nitrophenyl-
-D-galactoside as a substrate
(37).
PT-PCR analysis.
Confluent C38 and IB3 cells were stimulated with human TNF- and
anti-asialoGM1 antibody. Total RNA was isolated by ethanol precipitation and treated with DNase I before RT-PCR. Each RT reaction
was carried out by combining 5-10 µl of total RNA mix with 2 µl 10× RT buffer, 3 µl dNTP mix, 2 µl random primer, 1 µl
Moloney murine leukemia virus RT, and 1 µl RNase inhibitor (42°C
for 1 h). The PCR reaction was performed by combining 10 µl
cDNA, 2 µl primer mix (1 µl of each 5' and 3' primer), 5 µl 10×
buffer, 1 µl 50× Taq DNA polymerase, and 5 µl 10× dNTPs.
Conditions for PCR consisted of incubation at 94°C for 3 min; 30 cycles each of 94°C for 30 s, 60°C for 30 s, and 72°C
for 30 s, and incubation at 72°C for 7 min. Samples were
analyzed by gel electrophoresis on a 1.5% agarose gel. PCR primers for
human IL-8 were 5'-TACTCCAAACCTTTCCAACCC-3' and
5'-AACTTCTCCACAACCCTCTG-3'.
-Actin was used as a normalization marker.
Electrophoretic mobility shift assays.
Nuclear extracts were prepared by the method of Dignam et al.
(13) with some modifications. Electrophoretic mobility
shift assays were performed using nuclear extracts (4 µg) and binding buffer containing 5 mM Tris · HCl (pH 7.5), 37.5 mM KCl, 0.5 mM EDTA, 2% Ficoll, 50 µg/ml poly (dI-dC), and
30-100,000 cpm of -32P-labeled probe, and incubated
on ice for 15 min. Nuclear extracts were added and the mixture was
incubated at room temperature for 15 min. In some instances, antibodies
against either p65 RelA or p50, the NF-
B family of binding proteins,
were added (10 min at room temperature). The protein-DNA complexes were
analyzed by electrophoresis through a 5% polyacrylamide gel. The gels
were dried and exposed to radiographic film.
IKK in vitro phosphorylation assays.
Endogenous IKK was immunoprecipitated from cell extracts with an
anti-IKK- antibody (34). Activity of the immune complex was assayed in 30 µl of kinase buffer in the presence of 10 µM ATP,
5 µCi [
-32P] ATP, and GST-I
B
(3 µg/sample)
as a substrate (30°C for 15 min). Reactions were terminated with 4×
Laemmli sample buffer. Samples were resolved by 10% SDS-PAGE, and
the proteins were transferred to a nitrocellulose membrane by semidry
transfer. After Ponceau staining, the membrane was exposed to film, and
substrate phosphorylation was assessed by optical scanning. Equal
levels of immunoprecipitated IKK were confirmed by immunoblotting using
an anti-IKK antibody.
MAP kinase in vitro phosphorylation assays. Cells were transfected with cDNAs encoding HA-tagged p42 ERK2 or p38 MAP kinase and empty vector. In selected experiments, cells were cotransfected with MEKK1-KM or empty vector. The epitope tag was immunoprecipitated from cell extracts with an anti-HA monoclonal antibody, and an in vitro kinase assay was performed using recombinant MBP or ATF-2 as substrates, as described (36).
Data analysis.
Each experiment was performed at least three times. Data were described
as means ± SE. The significance of changes in luciferase activity
and protein abundance was assessed by analysis of variance. Differences
identified by analysis of variance were pinpointed by
Student-Newman-Keuls multiple range test. For reporter assays, changes
in promoter activity were calculated as arbitrary light units per
-galactosidase calorimetric units per hour and reported as fold
increase vs. baseline.
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RESULTS |
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Treatment of C-38 and IB3 cells with TNF- and anti-asialoGM1
induces IL-8 mRNA and protein expression.
C-38 and IB3 cells were treated with either TNF-
(10 ng/ml) or
anti-asialoGM1 (1:50) overnight, total mRNA was isolated, and RT-PCR
was performed. Treatment with TNF-
and anti-asialoGM1 each increased
IL-8 mRNA expression (Fig.
1A). However,
asialoGM1-stimulated IL-8 mRNA was increased in IB3 cells relative to
C-38 cells, consistent with the greater density of asialoGM1 receptors
in CF phenotype cells (5, 7, 20).
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NF-B binding in C-38 and IB3 cells.
To determine whether anti-asialoGM1 induces the binding of NF-
B to
DNA, nuclear extracts from treated cells were incubated with an
oligonucleotide encoding the consensus NF-
B binding site. Incubation
of C-38 and IB3 cells with a 1:50 dilution of anti-asialoGM1 induced
substantial NF-
B binding (Fig. 2, A and
B). AsialoGM1-induced NF-
B
binding was significantly increased in IB3 cells compared with C38
cells. Coincubation of nuclear extracts with antibodies against p65
RelA and p50 NF-
B1, but not c-Rel and RelB induced supershift of the
DNA binding complex, demonstrating the presence of these NF-
B family
transcription factors.
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IKK activation in C-38 and IB3 cells.
To examine whether IKK is activated in IB3 and C-38 cells, we measured
endogenous IKK activity by immunoprecipitating cell lysates with an
antibody against IKK- and incubating the precipitates with
[
-32P]ATP and recombinant I
B
as a substrate.
IKK-
functions as a regulatory subunit for the IKK complex and
serves as a docking site for upstream activators (21, 22).
AsialoGM1-induced responses were significantly increased in IB3
cells relative to C-38 cells (Fig. 3, A and
B).
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Requirement of IKK- for NF-
B transactivation and IL-8
promoter activity in IB3 cells.
To test whether IKK-
activation is required for transactivation of
NF-
B and transcription from the IL-8 promoter, cells were
cotransfected with cDNAs encoding either dominant negative IKK-
(IKK-
-AA) or empty vector and either NF-
B or IL-8 reporter plasmids. Luciferase activity was measured with a luminometer, and
results were normalized for transfection efficiency by measurement of
-galactosidase activity. Expression of IKK-
-AA attenuated anti-asialoGM1-induced NF-
B transactivation (Fig.
4A) and IL-8 promoter activity
(Fig. 4B). These results indicate that, in IB3 cells,
maximal asialoGM1-mediated NF-
B transactivation and transcription from the IL-8 promoter are each dependent on IKK-
activation.
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Requirements of NIK and MEKK1 for NF-B transactivation and IL-8
promoter activity in IB3 cells.
To determine the upstream signaling intermediates responsible for
IKK-
activation, IB3 cells were cotransfected with kinase-deficient NIK (NIK-KM), kinase-deficient MEKK1 (MEKK1-KM), or empty vector and
NF-
B and IL-8 reporter plasmids. NIK-KM attenuated both
anti-asialoGM1-induced NF-
B transactivation and IL-8 promoter
activity (Fig. 5, A and B), but MEKK1-KM had no effect
(Fig. 5, C and D). Furthermore, NIK-KM attenuated
both TNF-
and anti-asialoGM1-induced IKK-
activity (Fig.
5E). Finally, an in vitro kinase assay assessing the effect
of MEKK1-KM on p38 MAP kinase activity confirmed the inhibitory effect
of this mutant protein on kinase activity (Fig. 5F).
Together, these data suggest that NIK but not MEKK1 is an upstream
activator of IKK-
, NF-
B, and IL-8 in IB3 CF phenotype cells.
|
Requirement of ERK signaling for IL-8 expression but not NF-B
activation.
We examined the contribution of ERK signaling to asialoGM1-induced IL-8
expression. We measured ERK2 activation in IB3 and C-38 cells by in
vitro phosphorylation assay using MBP as a substrate. Ligation of
asialoGM1 induced ERK activation in both cell types (Fig.
6A). Consistent with previous
results, the level of ERK activation appeared greater in IB3 cells.
Pretreatment with a chemical inhibitor of MEK, U0126, reduced ERK
activation (Fig. 6A). A concentration of 3 µM U0126 was
sufficient for this response and was used for subsequent experiments.
Pretreatment with 3 µM U0126 also attenuated IL-8 protein abundance
(Fig. 6B) and transcription from the IL-8 promoter (Fig.
6C). Inhibition of ERK signaling by expression of a dominant
negative form of MEK1 also attenuated asialoGM1-induced transcription
in IB3 cells (Fig. 6D).
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DISCUSSION |
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Investigators have compared cytokine expression in airway
epithelial cells derived from CF individuals with those derived from
normal individuals. In at least three cell line pairs, IL-8 expression
has been found to be increased in CF phenotype cells relative to
corrected cells (15, 24). Further studies examining the
mechanism by which cytokine expression is upregulated in these cells
have demonstrated a potential role for NF-B signaling (14, 46,
48). However, the upstream intermediates responsible for P. aeruginosa-induced NF-
B responses in CF cells have not
been studied. To accomplish this, we examined the responses to ligation of the P. aeruginosa pilin receptor,
gangliotetraosylceramide (asialoGM1) in IB3 cells, an
adeno-associated virus-transformed human bronchial epithelial cell line
derived from a CF patient (
F508/W1282X), compared with its
CFTR-corrected line, C-38 cells. In the present study, we found that
ligation of the asialoGM1 receptor with specific antibody induced
greater IL-8 expression in IB3 cells than C-38 cells, consistent with
the greater density of asialoGM1 receptors in CF phenotype cells
(5, 7, 20). As in previous studies (14, 48),
asialoGM1-mediated activation of NF-
B was also greater in IB3 cells.
The basic NF-B complex is a dimer of two members of the Rel family
of proteins, p50 (NF-
B1) and p65 (RelA). In unstimulated cells,
NF-
B is sequestered in the cytoplasm by I
B proteins. Phosphorylation and degradation of I
B allow translocation of NF-
B
to the nucleus, where it regulates gene transcription by binding to
specific sequences of DNA. Although I
B
is the best characterized
family member, I
B
and I
B
may also regulate NF-
B (21). The "classical" signaling pathway to NF-
B
activation includes the successive phosphorylation and activation of
NIK and IKK (21, 22). IKK consists of two catalytic
subunits (IKK-
and IKK-
) and a regulatory subunit (IKK-
).
Although IKK-
and IKK-
contain similar kinase domains with
essentially identical activation loops (33), they are
functionally distinct. Recent studies suggest that IKK-
serves as
the target for proinflammatory signals, whereas IKK-
plays a
critical role in development (6, 9, 19). On the other
hand, it has recently been shown that UV-C irradiation (2,
29), hepatitis B protein X (38), and p21-activated
kinase (17) may each activate NF-
B via as yet unidentified IKK-independent mechanisms. In the present study, we found
that ligation of the asialoGM1 receptor with specific antibody induced
greater IKK activation in IB3 than C-38 cells, consistent with the
notion that IKK is a downstream target of the asialoGM1 receptor in CF
phenotype cells. Furthermore, expression of a dominant negative form of
IKK-
(IKK-
-AA) significantly attenuated asialoGM1-mediated
NF-
B transactivation and IL-8 promoter activity, strongly suggesting
that IKK-
is required for maximal asialoGM1-mediated I
B phosphorylation.
The most potent IKK activator yet identified is the serine-threonine
kinase NIK. NIK was originally identified as a protein that interacts
with TNF receptor-associated factor-2 (TRAF2). Constitutive activation
of NIK causes IB
degradation and NF-
B activation
(31), suggesting that NIK is involved in NF-
B
signaling. NIK is required for activation of NF-
B by non-typeable
Hemophilus influenzae (44) and micrococci
(47). On the other hand, NIK strongly and preferentially
interacts with and activates IKK-
rather than IKK-
(30,
49). In addition, replacement of the COOH-terminal TRAF domain
of TRAF2 or TRAF6 with a heterologous oligodimerization domain results
in chimeric proteins that retain the ability to activate IKK, although
they no longer interact with NIK (1).
IKK- and the MAP kinase kinases (MKKs) share structural elements,
including the position of two serine phosphoaccepting sites within the
activation loop. Accordingly, it was later found that MKK kinases,
including those of the MAP kinase/MEKK family, may phosphorylate and
activate IKK (34). MEKK1 is activated by TNF-
and IL-1,
and overexpression of a dominant negative MEKK1 inhibits IKK-
and
NF-
B activation (26, 34). Other putative IKK kinases include protein kinase C-
(25) and Akt/protein kinase B
(35, 41). To determine the requirement of NIK and MEKK1
for NF-
B activation and IL-8 expression in CF cells, we transiently
transfected cells with cDNAs encoding kinase-inactive mutants of NIK
and MEKK1. We found that inhibition of NIK but not MEKK1 attenuated
TNF-
and asialoGM1-mediated NF-
B and IL-8 responses, suggesting
that NIK is required for maximal IKK-
activation. Expression of
kinase-inactive NIK also inhibited basal and stimulated IKK-
activity, consistent with the notion that NIK is a major upstream
activator of IKK-
and NF-
B in CF airway cells.
With the use of a series of chemical inhibitors, Li and colleagues
(28) concluded that P. aeruginosa
lipopolysaccharide induces NCI-H292 pulmonary epithelial cell MUC2
expression via a Src/Ras/ERK/RSK/NF-B pathway. Similarly, Ratner and
colleagues (40), using a series of chemical inhibitors,
found that activation of NF-
B, ERK, and p38 was each required for
maximal IL-8 expression in 1HAEo
human airway epithelial
cells incubated with P. aeruginosa or antibody to asialoGM1.
However, the precise relationship between ERK activation and NF-
B
activation in CF epithelial cells has not been studied. We previously
found that in 16HBE14o
human bronchial epithelial cells,
ERK regulated IL-8 expression in an AP-1-dependent, NF-
B-independent
manner (27). In the present study, we found that although
ERK was required for maximal IL-8 expression, inhibition of ERK
signaling had no effect on IKK-
or NF-
B activation, confirming
that in human bronchial epithelial cells ERK regulates IL-8 expression
in an NF-
B-independent manner.
Finally, as noted previously (14, 46), we found that basal
levels of IL-8 expression and NF-B activation were modestly increased in IB3 cells relative to corrected C-38 cells. We did not
detect basal increases in endogenous IKK activity in IB3 cells, suggesting that IKK is not responsible for the observed differences in
NF-
B activation. However, it should be noted that basal levels of
IL-8 expression and NF-
B activation were relatively small compared
with asialoGM1-mediated responses, and therefore we did not choose to
investigate the underlying mechanism for these differences.
In summary, we showed that asialoGM1-mediated activation of IKK, NIK, and ERK is each increased in a phenotypic CF bronchial epithelial cell line, relative to the corrected line, consistent with the greater density of asialoGM1 receptors in CF phenotype cells (5, 7, 20). Inhibition of these kinases, each of which was required for maximal IL-8 expression, may attenuate the inflammatory response in CF respiratory epithelia.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. P. Zeitlin (Johns Hopkins University) for the contribution of IB3-1 and C-38 cells and Dr. A. Brasier (University of Texas, Galveston, TX), Dr. M. Karin (University of California, San Diego, CA), and Dr. M. Rosner (University of Chicago) for the gifts of plasmid vectors.
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FOOTNOTES |
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These studies were supported by National Institutes of Health Grants CA-73740 (A. Lin) and HL-56399 (A. Lin and M. B. Hershenson), American Cancer Society Grant CCG-98471 (A. Lin), and the Cystic Fibrosis Foundation (M. B. Hershenson).
Address for reprint requests and other correspondence: M. B. Hershenson, Univ. of Michigan, 1150 W. Medical Center Dr., 3570 MSRBII/Box 0688, Ann Arbor, MI 48109-0688 (E-mail: mhershen{at}umich.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.
First published September 27, 2002;10.1152/ajplung.00086.2002
Received 22 March 2002; accepted in final form 20 September 2002.
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