1 Department of Pediatrics, University of Chicago, Chicago, Illinois 60637; and 2 Department of Medicine, University of Texas Medical Branch, Galveston, Texas 77555-1060
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ABSTRACT |
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Recent studies indicate that maximal IL-8
protein expression requires activation of NF-B as well as activation
of the MAP kinases ERK, JNK, and p38. However, the precise relationship
between NF-
B transactivation and MAP kinase activation remains
unclear. We examined the requirements of NF-
B, ERK, JNK, and p38 for
TNF-
-induced transcription from the IL-8 promoter in a human
bronchial epithelial cell line. Treatment with TNF-
induced
activation of all three MAP kinases. Using a combination of chemical
and dominant-negative inhibitors, we found that inhibition of NF-
B,
ERK, and JNK, but not p38, each decreased TNF-
-induced transcription
from the IL-8 promoter. Inhibition of JNK signaling also substantially
reduced TNF-
-induced NF-
B transactivation, whereas inhibition of
ERK and p38 had no effect. On the other hand, ERK was required and sufficient for TNF-
-induced activation of activator protein (AP)-1 promoter sequences, which together function as a basal level enhancer. JNK activation was also required for AP-1 transactivation. Finally, inhibition of p38 attenuated IL-8 protein abundance, suggesting that
p38 regulates IL-8 expression in a posttranscriptional manner. We
conclude that, in human airway epithelial cells, MAP kinases may
regulate IL-8 promoter activity by NF-
B-dependent (in the case of
JNK) and -independent (ERK) processes, as well as by
posttranscriptional mechanisms (p38).
cytokines; inflammation; signal transduction; transcription factors; interleukin 8; mitogen-activated protein
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INTRODUCTION |
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AIRWAY EPITHELIAL CELLS SYNTHESIZE a number of cytokines including the neutrophil chemoattractant and activator interleukin (IL)-8 (19, 20, 75). IL-8 expression is increased in the airways of patients with asthma (1, 38, 51). Environmental factors that may alter airway reactivity, including viruses (15, 26, 43, 56, 58), allergens (47), cigarette smoke (63), and air pollutants (3, 11, 13, 19, 42), have each been demonstrated to increase airway or pulmonary epithelial cell IL-8 expression. Increased levels of IL-8 have been found in the bronchoalveolar lavage of infants developing bronchopulmonary dysplasia (44, 72). Finally, IL-8 is increased in the airways of patients with cystic fibrosis (7, 46), and exposure of cultured airway epithelial cells to Pseudomonas gene products has been noted to further increase IL-8 expression (17, 22). Together, these data suggest that airway epithelial cell IL-8 expression may play an important pathogenetic role in airways diseases such as asthma, bronchopulmonary dysplasia, and cystic fibrosis.
The transcription factor complex nuclear factor-B (NF-
B) appears
to play a key role in the regulation of lung epithelial cell cytokine
expression (5). The basic NF-
B complex is a dimer of
two members of the Rel family of proteins, p50 (NF-
B1) and p65 (Rel
A). Both subunits contact DNA, but only Rel A contains a
transactivation domain near its COOH terminus that directly interacts
with the basal transcription apparatus. In unstimulated cells, NF-
B
is sequestered in the cytoplasm by I
B proteins. However,
phosphorylation and degradation of I
B allow translocation of NF-
B
to the nucleus, where it may regulate gene transcription by binding to
specific sequences of DNA. I
B has been demonstrated to be
phosphorylated by I
B kinase (IKK), which may be phosphorylated in
turn by a number of kinases including NF-
B-inducing kinase, mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) kinase (MEKK), and protein kinase C-
(45, 50, 52,
81). In A549 type II pulmonary epithelial cells,
pretreatment with the proteasome inhibitor MG-132, which prevents I
B
degradation, has been demonstrated to reverse the effects of TNF-
on
NF-
B binding and IL-8 in these cells (25). The airway
epithelium of patients with asthma demonstrates increased translocation
and DNA binding of the NF-
B subunit p65 Rel A (31), and
treatment of patients with budesonide decreases NF-
B DNA binding
activity (30). Together, these data demonstrate the
importance of NF-
B for pulmonary epithelial cell IL-8 expression.
The transcriptional regulation of IL-8 expression in lung epithelial
cells involves not only NF-B but also activator protein (AP)-1 and
NF-IL-6 (C/EBP
) promoter sequences (26, 49, 55, 57).
Activation of MAP kinases, which may translocate from the cytoplasm to
the nucleus after mitogenic stimulation, has been shown to induce
phosphorylation and increase the trans-activating activity
of a number of nuclear transcription factors, including the AP-1
transcription factors c-Fos and c-Jun (4, 18, 29, 35, 67,
77) and C/EBP
(39). Accordingly, recent studies have shown that MAP kinases may regulate IL-8 expression. On the basis
of chemical inhibitor studies, ERK activation has been demonstrated to
be required for IL-8 mRNA or protein expression in THP-1 human monocytic leukemia cells (54), A549 lung epithelial cells
(14), squamous cell carcinoma cell lines (2),
and gastric cancer cells (64). Using genetic inhibitors,
investigators have shown JNK activation to be required for IL-8 mRNA or
protein expression in human embryonic kidney cells (37),
the human epidermal carcinoma KB cell line (48), and the
OVCA 420 human ovarian cancer cell line (53). Inhibition
of p38 activation using chemical inhibitors has been demonstrated to
reduce IL-8 mRNA and protein expression in human peripheral blood
mononuclear cells (70), neutrophils (83),
mast cells (24), intestinal epithelial cells
(36), pulmonary vascular endothelial cells
(32), and lung epithelium-like H292 cells (28, 33,
59).
Given the importance of AP-1 promoter sequences for transcription from
the IL-8 promoter (49, 57), the observed requirement of
MAP kinase activation for maximal IL-8 expression may relate to its
role in AP-1 transactivation. On the other hand, it is also possible
that cross talk occurs between the MAP kinases and NF-B. Maximal
NF-
B transactivation may require not only translocation to the
nucleus and assembly of the transcription complex, but additional
phosphorylation events via different MAP kinases as well. In murine
fibrosarcoma cells, the transcriptional activity of the nuclear NF-
B
complex appears to be regulated by a p38 MAP kinase-dependent
phosphorylation step involving a protein of the transactivation complex
(73). A recent study in a human acute monocytic leukemia
cell line (THP-1) demonstrated this protein to be the basal
transcription factor TATA-binding protein (10). In
addition, analysis of the IKK protein sequences reveals several potential phosphoacceptor sites in a region conserved in all protein kinases, the T (or activation) loop, that resemble those that are used
by MAP kinase kinase kinases to activate MAP kinase kinases (MKKs)
(45, 60). As noted above, it has been shown that MEKK isoforms may phosphorylate and activate IKK, specifically MEKKs 1-3 (52, 81). JNK1 has been shown to interact with
c-Rel in Jurkat T cells (61). Finally, a phosphorylation
target of ERK, the 90-kDa ribosomal S6 kinase (6),
phosphorylates the NH2-terminal regulatory domain of
I
B
and stimulates its degradation (27, 69).
In this study, we examine the precise contributions of MAP kinase
activation and NF-B transactivation to human airway epithelial cell
IL-8 expression. We found that inhibition of ERK, JNK, and NF-
B, but
not p38, each decreases TNF-
-induced transcription from the IL-8
promoter. Inhibition of JNK signaling also substantially reduced
TNF-
-induced NF-
B activation, whereas inhibition of ERK and p38
had no effect. On the other hand, ERK was required (and sufficient) for
TNF-
-induced activation of AP-1 promoter sequences, which function
as a basal level enhancer. JNK activation was also required for AP-1
transactivation. Finally, inhibition of p38 attenuated IL-8 protein
abundance, suggesting a posttranscriptional effect on IL-8 protein
expression. These data suggest that, in human airway epithelial cells,
MAP kinases may regulate IL-8 promoter activity by NF-
B-dependent
(in the case of JNK) and -independent (ERK) processes, as well as by
posttranscriptional mechanisms (p38).
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EXPERIMENTAL PROCEDURES |
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Cell culture.
A derivative of 16HBE14o human bronchial epithelial
cells, provided by S. White (University of Chicago), was studied. Cell lines were originally established from bronchial epithelial tissue by
transfection with pSVori
, which contains the
origin-defective SV40 genome (16). Unlike the parental
line, these cells do not grow in distinct clusters and demonstrate
improved transfection efficiency. Cultures show specific immunostaining
with pancytokeratin c11 antibody (Santa Cruz Biotechnology, Santa Cruz,
CA), bind galactose or galactosamine-specific lectins particular to
basal epithelial cells (23), and express
1-,
2-,
3- and
6-integrin subunits on their cell surface
(76). Cells were grown on coated plates (10 µg/ml
fibronectin, 30 µg/ml collagen, and 100 µg/ml bovine serum albumin)
in Eagle's minimum essential medium (MEM) with 10% fetal bovine serum
(FBS), 1% penicillin-streptomycin, and 200 mM L-glutamine.
Plasmid vectors.
The 162/+44 fragment of the full-length human IL-8 promoter was
subcloned into a luciferase reporter plasmid (
162/+44 hIL-8/Luc). The
reporter activities of this fragment have been shown to be identical to
the full-length promoter in response to respiratory syncytial virus
infection (26), and this fragment contains the NF-
B,
nuclear factor for IL-6 (NF-IL-6), and AP-1 binding sites required for
maximal TNF-
responses (8). Site-directed mutagenesis of the AP-1 site in the context of the
162/+44 hIL-8 was introduced by polymerase chain reaction with mutagenic primers to obtain
AP-1
162/+44 hIL-8/Luc (26). A hemagglutinin-tagged ERK2
(pCDNA3-HA-ERK2) was constructed by ligating a DNA fragment encoding
the seven-amino acid influenza hemagglutinin epitope to the 5'-end of
murine ERK2 (34). Plasmid DNAs encoding pCMV-LacZ and
hemagglutinin-tagged forms of JNK1 (62) and p38
(79) were provided by M. Rosner (University of Chicago).
Plasmids encoding dominant-negative (pCMV-MEK-2A) and constitutively
active (pCMV-MEK-2E) forms of MAP kinase/ERK kinase (MEK1), in which
serine-218 and -222 phosphorylation sites were modified to alanine or
glutamic acid, respectively, were provided by D. Templeton (Case
Western Reserve University) (80). A plasmid
encoding kinase-inactive MKK7 (pSR
3-JNKK2-KM), in which Lys-149 in
the ATP-binding domain was replaced by methionine, was provided by A. Lin (University of Chicago) (82). NF-
B and AP-1
reporter plasmids (NF-
B-TATA/Luc and AP-1-TATA/Luc, respectively) were purchased from Stratagene. A cDNA encoding a nonphosphorylatable I
B mutant in which the NH2-terminal 36 amino acids were
deleted, including critical serine-32 and -36 phosphorylation sites
(pCMV4-I
B
N), was provided by D. Ballard (Vanderbilt University)
(9). GST-Jun (1-79) was obtained from J. Posada (University of Vermont) (71).
Chemical inhibitors.
The ERK inhibitor U-0126 was obtained from Promega (Madison, WI). The
p38 MAP kinase inhibitor SB-202190 was obtained from Calbiochem (San
Diego, CA). Cells were incubated with chemical inhibitors 60 min before
treatment with TNF-.
Reporter assays.
To measure transcription from the IL-8 promoter or its site-directed
mutants, or NF-B or AP-1 transactivation, we transfected cells with
the relevant reporter plasmid using a liposome-mediated technique. To
examine the effect of genetic inhibitors or activators, we
cotransfected cells with cDNA encoding either empty vector or the
mutant protein of interest, as described (68).
Transfection efficiency was assessed by cotransfection with pCMV-LacZ.
After 8 h of serum starvation, cells were treated with TNF-
(Upstate Biotechnology, Lake Placid, NY). In selected experiments,
cells were pretreated for 60 min with U-0126 or SB-202190. Sixteen
hours after treatment, cells were harvested and analyzed for luciferase and
-galactosidase activities, as described (66, 68).
Electrophoretic mobility shift assays.
Nuclear extracts were prepared by the method of Dignam et
al. (21) 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 counts/min 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 p65 Rel
A, p50 NF-
B1, c-Rel, Rel B, c-Jun, or c-Fos were added (10 min at
room temperature; all antibodies from Santa Cruz Biotechnology, Santa Cruz, CA). Oligonucleotide probes encoding the consensus
sequences of NF-
B and AP-1 family transcription factors were
purchased from Promega. The protein-DNA complexes were analyzed by
electrophoresis through a 5% polyacrylamide gel. The gels were dried
and exposed to radiographic film.
Measurement of MAP kinase activities.
To examine ERK, JNK, and p38 activities, we transfected cells with cDNA
encoding a hemagglutinin-tagged form of ERK2, JNK1, or p38 and
either empty vector or MEK-2A, MEK-2E, or JNKK2-KM, as described
(65, 68). Forty-eight hours after transfection, cells were
serum starved in MEM. The next day, cells were treated with TNF-
or
10% FBS. In selected experiments, cells were pretreated for 60 min
with U-0126 or SB-202190. Activation of MAP kinases was assessed by
immunoprecipitation of the epitope tag using the mouse monoclonal
anti-hemagglutinin antibody HA.11 (Babco, Richmond, VA) followed by an
in vitro phosphorylation assay using myelin basic protein (MBP; Sigma,
St. Louis, MO), c-Jun, or ATF-2 (New England Biolabs, Beverly, MA) as
substrates (65). To confirm the expression of
hemagglutinin-tagged ERK2, JNK1, or p38
in airway epithelial cell
immunoprecipitates, we probed nitrocellulose membranes with HA.11
anti-hemagglutinin. Signals were amplified and visualized using
peroxidase-linked rat anti-mouse
-light chain IgG (Zymed
Laboratories, South San Francisco, CA) and enhanced chemiluminescence.
Measurement of IL-8 protein.
Cells were serum starved for 24 h and then treated with TNF-
overnight. Conditioned media were collected, centrifuged to remove cell
debris (14,000 rpm for 10 min), and frozen at
80°C. IL-8 protein
was measured by ELISA (Amersham Life Science, Arlington Heights, IL).
Data analysis.
Each experiment was performed at least three times. Statistical
significance was assessed by analysis of variance (ANOVA). Differences
identified by ANOVA 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 h.
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RESULTS |
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TNF- treatment of human airway epithelial cells induces IL-8
expression.
To test whether TNF-
induces transcription from the IL-8
promoter in human airway epithelial cells, we transfected SV40 T antigen-transformed human airway epithelial cells
(16HBE14o
cell line) with cDNA encoding the full-length
human IL-8 promoter subcloned into a luciferase reporter plasmid. Cells
were transfected with a liposome solution, as described
(68). Sixteen hours after TNF-
treatment, cells were
lysed, and luciferase activity was measured with a luminometer. TNF-
induced IL-8 promoter activity in a concentration-dependent manner
(Fig. 1A). To determine
whether changes in promoter activity were reflected in protein
expression, we incubated cells with TNF-
(5 ng/ml) overnight.
Aliquots of conditioned medium were examined for IL-8 protein by ELISA.
TNF-
treatment induced an almost 10-fold increase in IL-8 protein
abundance (Fig. 1B).
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NF-B activation is required for transcription from the IL-8
promoter in human airway epithelial cells.
To determine whether TNF-
induces the binding of NF-
B to DNA, we
incubated nuclear extracts from treated cells with an oligonucleotide encoding the consensus NF-
B binding site. Incubation of airway epithelial cells with 10 ng/ml TNF-
induced significant NF-
B binding (Fig. 2A).
Furthermore, co-incubation of nuclear extracts with an antibodies
against p65 Rel A and p50 NF-
B1 each induced a supershift of the DNA
binding complex, demonstrating the presence of these NF-
B family
transcription factors. Incubation with antibodies against c-Rel and Rel
B or the AP-1 transcription factors c-Jun and c-Fos (not shown) had no
effect on the protein-DNA complexes. To determine whether TNF-
treatment induces transactivation of NF-
B promoter sequences, we
cotransfected cells with a cDNA encoding a series of NF-
B consensus
binding sequences linked to a minimal promoter and luciferase
(NF-
B-TATA/Luc). As expected, 10 ng/ml TNF-
treatment
induced substantial NF-
B transactivation (Fig. 2B). To
test whether NF-
B activation is required for transcription from the
IL-8 promoter, we transfected human airway epithelial cells with the
IL-8 reporter construct and a cDNA encoding a nonphosphorylatable, NH2-terminal deletion mutant of I
B (I
B
N), the
phosphorylation and degradation of which are required for NF-
B
activity (9). Expression of the NH2-terminal
mutant, but not a normally functioning COOH-terminal mutant,
attenuated IL-8 promoter activity (Fig. 2C). These results
indicate that IL-8 promoter activation is largely dependent on
NF-
B.
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Requirement of MAP kinases for TNF--induced transcription from
the IL-8 promoter.
We tested whether TNF-
treatment induces activation of MAP kinases.
Cells were transfected with hemagglutinin-tagged forms of ERK2, JNK1,
or p38
and treated with TNF-
(10 ng/ml). Cell lysates were
immunoprecipitated with an antibody against the epitope tag, followed
by in vitro phosphorylation using the appropriate substrate (Fig. 3,
A-C). Relative to the effects of phorbol ester, treatment with TNF-
induced modest ERK2 activation, as shown by
phosphorylation of MBP (Fig.
3A). TNF-
induced
substantial activation of JNK1 and p38
, as shown by phosphorylation
of c-Jun (Fig. 3B) and ATF-2 (Fig. 3C),
respectively. In vitro phosphorylation assays confirmed the inhibitory
effects of U-0126, MEK-2A, JNKK2-KM, and SB-202190 on ERK2, JNK1, and
p38
activities (Fig. 3, A-D).
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TNF--induced NF-
B activation is independent of ERK but
dependent on JNK activity.
To determine whether ERK is upstream of NF-
B transcriptional
activation, we examined the effects of U-0126 on TNF-
-induced NF-
B DNA binding or transactivation. In these experiments, U-0126 did not attenuate either NF-
B DNA binding or transactivation (Fig.
6, A and B),
suggesting that ERK was not required. These data suggest that, whereas
ERK and NF-
B are both required for TNF-
-induced transcription
from the IL-8 promoter, they do so via separate pathways. To determine
whether JNK might be an upstream activator of NF-
B transcriptional
activation, we cotransfected cells with NF-
B-TATA/Luc and a cDNA
encoding JNKK2-KM. Overexpression of JNKK2-KM reduced NF-
B-TATA/Luc
activity (Fig. 6C), suggesting that JNK functions as an
upstream activator of NF-
B or increases its transactivation. As with
transcription from the IL-8 promoter, chemical inhibition of p38 failed
to decrease TNF-
-induced NF-
B transactivation (Fig.
6D).
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MAP kinase activation and AP-1 transactivation.
Because inhibition of MEK reduced IL-8 but not NF-B reporter
activity, we asked whether ERK functions through IL-8 promoter AP-1
sequences. First, we tested whether TNF-
treatment induces binding
of AP-1 nuclear proteins to DNA. TNF-
treatment (10 ng/ml) induced
binding of nuclear proteins to an oligonucleotide encoding the AP-1
consensus binding sequence (Fig.
7A). Inhibition of MEK1 with
U-0126 attenuated binding, demonstrating that TNF-
-induced AP-1
activation is ERK dependent. Next, cells were cotransfected with
162/+44 hIL-8/Luc or a site-directed mutant of the IL-8 promoter AP-1
site (
AP-1
162/+44 hIL-8/Luc) (26) and either empty
vector or a cDNA encoding a constitutively active form of MEK1
(MEK-2E). Selected cultures were treated with TNF-
. Relative to
TNF-
, MEK1 activation induced modest but significant IL-8 promoter
activity (Fig. 7B). Mutation of the AP-1 site blocked basal
and MEK1-induced activation, although responsiveness to TNF-
was
maintained. These data suggest that the AP-1 site functions as a basal
level enhancer and that AP-1 promoter sequences are required for
ERK-mediated transcription. Using AP-1 (AP-1-TATA/Luc) and
NF-
B reporter plasmids, we found that activation of MEK1 was
sufficient for AP-1 but not NF-
B transactivation (Fig.
7C), consistent with the notion that ERK functions through
IL-8 promoter AP-1 sequences. Using the AP-1 reporter plasmid, we found
that chemical inhibition of MEK1 activation with U-0126 blocked
TNF-
-induced AP-1 activity, confirming that ERK activation is
required for transactivation of the AP-1 site by TNF-
(Fig.
7D). Finally, inhibition of JNK1 activation by expression of
JNKK2-KM also attenuated TNF-
-induced AP-1 transactivation (Fig.
7E).
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DISCUSSION |
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We have found in a human airway epithelial cell line that
1) overexpression of a nonphosphorylatable IB (I
B
N)
attenuates transcription from the IL-8 promoter, 2)
inhibition of ERK by either chemical inhibitor or a dominant-negative
MEK1 attenuates IL-8 promoter activity, 3) inhibition of JNK
signaling by overexpression of a dominant-negative MKK7
(JNKK2-KM) decreases transcription from the IL-8 promoter,
4) inhibition of p38 MAP kinase by pretreatment with a chemical inhibitor fails to reduce IL-8 promoter activity but
attenuates protein abundance, 5) inhibition of JNK but not ERK decreases NF-
B transactivation, and 6) ERK and JNK
are each required for TNF-
-induced AP-1 transactivation. These
findings are discussed below.
The importance of NF-B promoter sequences for transcription from the
IL-8 promoter in lung epithelial cells has been well studied (26,
55, 57). In A549 type II pulmonary epithelial cells,
pretreatment with the proteasome inhibitor MG-132, which prevents I
B
degradation, has been demonstrated to reverse the effects of TNF-
on
NF-
B binding and IL-8 in these cells (25). In the
present study, we confirm that I
B phosphorylation is required for
TNF-
-induced responses. Because the MAP kinases have been shown to
regulate cytokine expression in a number of cell types, including lung
epithelial cells (14, 28, 32, 33, 59), we examined the
requirements of the ERK, JNK, and p38 for transcription from the IL-8
promoter in airway epithelial cells and questioned whether these
signaling intermediates function via activation of NF-
B.
We found that inhibition of ERK by either pretreatment with a chemical
inhibitor or expression of a dominant-negative MEK1 each attenuated
TNF--induced transcription from the IL-8 promoter. These data
confirm a previous report in THP-1 human monocytic leukemia cells,
demonstrating that ERK may regulate IL-8 expression on the
transcriptional level (54). We now demonstrate that
inhibition of ERK has no effect on NF-
B binding to DNA or
transactivation, suggesting that ERK regulates IL-8 expression in an
NF-
B-independent manner. Consistent with this notion, activation of
ERK by overexpression of a constitutively active MEK1 did not induce
NF-
B transactivation. These data extend previous findings by
Janssen-Heininger and colleagues (41), who showed that
expression of a dominant-negative Ras, an upstream activator of ERK,
failed to attenuate TNF-
-induced NF-
B transactivation in rat lung
epithelial (RLE) alveolar type II cells.
Activation of ERK signaling has been shown to induce phosphorylation
and increase the trans-activating activity of the AP-1 transcription factors c-Fos and c-Jun (4, 29, 67, 77). Given the importance of AP-1 promoter sequences for transcription from
the IL-8 promoter (49, 57), we assessed the requirement of
AP-1 promoter sequences for TNF--induced transcription from the IL-8
promoter, the requirement of ERK for AP-1 binding and transactivation,
and the sufficiency of ERK activation for AP-1 transactivation.
Inhibition of MEK1 with U-0126 attenuated AP-1 binding and
transactivation, demonstrating that TNF-
-induced AP-1 activation is
ERK dependent. Furthermore, mutation of the IL-8 promoter AP-1 site
blocked basal and MEK1-induced activation, whereas the response to
TNF-
was partially maintained. Finally, overexpression of active
MEK1 was sufficient for AP-1 but not NF-
B transactivation. Together,
these data suggest that ERK regulates transcription from the IL-8
promoter by activating the AP-1 site, which, in the context of TNF-
treatment, functions as a basal level enhancer. Studies examining the
IL-8 promoter AP-1 mutant have noted this function previously
(12, 74). We extend these findings by noting that ERK is
required and sufficient for AP-1 transactivation and DNA binding.
We also found that inhibition of JNK signaling by
overexpression of a dominant-negative MKK7 (JNKK2-KM)
decreases transcription from the IL-8 promoter. Furthermore, inhibition
of JNK signaling attenuated both NF-B and AP-1 transactivation. As
far as we are aware, this is the first demonstration that JNK is
required for IL-8 expression in lung cells. Although we did not confirm
the requirement for JNK with another inhibitor, the relative reduction in IL-8 promoter activity observed after pretreatment with high concentrations of SB-202190 is consistent with this finding, as high
concentrations of this chemical inhibitor have been shown to block both
p38 MAP kinase and JNK (40).
To examine the requirement of JNK for NF-B transactivation, we
overexpressed JNKK2-KM and measured the luciferase activity of a
NF-
B reporter plasmid. JNKK2-KM significantly reduced
TNF-
-induced NF-
B transactivation. Together with experiments
showing that overexpression of I
B
N attenuates IL-8 promoter
activity, these data suggest that inhibition of JNK signaling
attenuates IL-8 expression by reducing transactivation of the NF-
B
transcription factor complex. This notion is consistent with the work
by Janssen-Heininger and colleagues (41), in which
overexpression of JNK1 and 2 in RLE alveolar type II cells enhanced
oxidant-induced NF-
B transactivation. JNK1 has been shown to
interact with c-Rel in Jurkat T cells (61). However, as we
did not identify c-Rel in NF-
B binding protein complexes, the
mechanism by which JNK regulates NF-
B in our system remains unknown.
Finally, it is conceivable that JNKK2-KM could attenuate NF-
B
signaling by titrating its upstream activator MEKK1, which has been
demonstrated to activate NF-
B via IKK
.
Recent studies using the pyridinylimidazole compound SB-203580 have
demonstrated that p38 MAP kinase is required for IL-8 mRNA and protein
expression in lung epithelium-like H292 cells (28, 59). In
the present study, we confirmed that p38 activation is required for
IL-8 protein expression. However, inhibition of p38 did not inhibit
either NF-B transactivation or IL-8 promoter activity, suggesting
that, in human bronchial epithelial cells, p38 regulates IL-8
expression in a posttranscriptional manner. Although these data are in
conflict with earlier studies in H292 cells, p38 has been previously
shown to regulate IL-8 protein abundance by increasing the stability of
IL-8 mRNA (37, 78). It is therefore likely that the
inhibitory effect of SB-202190 we observed on IL-8 protein relates to
inhibition of translation, rather than transcription. It should also be
noted that smaller concentrations of SB-202190 appeared to increase
transcription from the IL-8 promoter, suggesting that p38 may also
function to inhibit transcription from the IL-8 promoter.
In conclusion, we have found in a human airway epithelial cell line
that inhibition of the ERK, JNK, and p38 MAP kinases attenuates IL-8
expression, albeit by different mechanisms. ERK and JNK regulate IL-8
promoter activity by NF-B-independent and NF-
B-dependent processes, respectively, whereas p38 regulates IL-8 expression by
posttranscriptional mechanisms. Given the potential pathogenetic role
of IL-8 expression in asthma, bronchopulmonary dysplasia, and cystic
fibrosis (1, 7, 38, 44, 46, 51, 72), these data suggest
that signaling intermediates of the ERK, JNK, and p38 pathways may
represent important targets for therapeutic intervention in airways diseases.
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ACKNOWLEDGEMENTS |
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The authors sincerely thank Marsha Rosner, Dennis Templeton, Anning
Lin, Dean Ballard, and James Posada for gifts of plasmid vectors and
Steven White for his gift of 16HBE14o human bronchial
epithelial cells.
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FOOTNOTES |
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* J. Li and S. Kartha contributed equally to this work.
These studies were supported by National Heart, Lung, and Blood Institute Grant HL-56399 and by the Cystic Fibrosis Foundation.
Address for reprint requests and other correspondence: M. B. Hershenson, Univ. of Chicago Children's Hospital, 5841 S. Maryland Ave., MC4064, Chicago, IL 60637-1470 (E-mail: mhershen{at}midway.uchicago.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.
March 15, 2002;10.1152/ajplung.00060.2002
Received 13 February 2002; accepted in final form 7 March 2002.
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