From INSERM U. 467, CHU Necker, 156 rue de Vaugirard,
75015 Paris and ¶ INSERM U. 412, Hôpital Trousseau, CHU
Saint Antoine, 26, av. du Dr Netter, 75012 Paris, France
Received for publication, July 25, 2000, and in revised form, November 2, 2000
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ABSTRACT |
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Inflammation of the airways is a major feature of
the inherited disease cystic fibrosis. Previous studies have shown that the pro-inflammatory cytokines tumor necrosis factor Cystic fibrosis (CF)1 is
an inherited multisystem disease caused by a number of mutations that
affect the gene encoding the cystic fibrosis transmembrane conductance
regulator (CFTR) (1-3), an integral membrane protein that functions as
a cAMP-regulated chloride channel (4). Despite our increased
understanding of the structure and function of CFTR, the mechanisms by
which the absence or dysfunction of CFTR causes numerous disorders is
less well documented. One of these puzzling disorders is the
destruction of the lung by infective-inflammatory injury, which is the
most common cause of morbidity in cystic fibrosis. Several clinical studies of the airways of young patients with cystic fibrosis have
found excessive amounts of pro-inflammatory cytokines, even in the
absence of any clinical lung disease or detectable infection (5). At
least two of these pro-inflammatory peptides, TNF NF- Previous studies on CFTR transcription have shown that the
cell specificity and the low concentration of CFTR are at least partly
dictated by the genomic sequences 5' upstream of the transcription initiation region (9, 10). Analysis of the CFTR 5'-region revealed that the CFTR promoter is a TATA-less promoter,
which probably explains why transcription is initiated at multiple
start points (9-11) and has a high G/C content. Studies on the
CFTR promoter have provided differing results on the
location of the major transcription initiation site and the minimal
promoter length (9, 11). This proximal region of the CFTR
promoter contains many GC boxes that could be SP1 binding sites (9) and
a cAMP-response element (CRE) at We have therefore investigated the effect of IL-1 Cell Culture and Reagents--
Calu-3, HT-29, and T84 cells were
obtained from the ATCC (American Type Culture Collection, Rockville,
MD); Calu-3 and HT-29 cells were cultured at 37 °C,
5%CO2 in Dulbecco's modified Eagle's medium and T84
cells in Dulbecco's modified Eagle's medium/F-12 medium (Life
Technologies, France). The medium for Calu-3 cells was supplemented
with nonessentials amino acids, pyruvate and HEPES. All media contained
antibiotics (penicillin, streptomycin, 50 µg/ml each) and 10% fetal
calf serum (Life Technologies). IL-1 Northern Blot Analysis--
Aliquots (15 µg) of total RNA
isolated from Calu-3 or T84 cells using the TRIzol reagent (Life
Technologies) were size-fractionated by agarose gel electrophoresis.
The RNA was then transferred to a nylon membrane (Promega,
Charbonnières, France) by capillary blotting, fixed by heating,
and hybridized under standard conditions with the Quick Hyb protocol
provided by Stratagene (Ozyme, Les Ulis, France). The
32P-labeled CFTR probe consisted of the 1.5-kb
EcoRI-EcoRI fragment of human CFTR
cDNA labeled by random priming. The membrane was also hybridized
with a human Nuclear Extracts--
Cells were rinsed twice with cold
phosphate-buffered saline (PBS), pH 7.4, scraped off into cold PBS, and
centrifuged at 600 × g for 3 min at 4 °C. The
resulting cell pellet was suspended in lysis buffer A (10 mM HEPES-NaOH (pH 7.9), 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM
dithiothreitol (DTT), 0.5 mM EDTA, and supplemented with an
antiprotease mixture (Roche Molecular Biochemicals). IGEPAL CA 630 (Sigma) detergent was then added (0.05%, v/v), and the cells were left
on ice for 10 min. The crude nuclei released by lysis were pelleted by
centrifugation at 1100 × g for 10 min at 4 °C,
washed in lysis buffer A, and suspended in buffer C (20 mM
HEPES (pH 7.9), 25% (v/v) glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.5 mM DTT, 0.5 mM EDTA and antiprotease mixture (Roche Molecular Biochemicals) by vigorous pipetting. The lysis of nuclei was checked under a phase-contrast microscope. The nuclei were left on ice for 15 min, vortexed, and clarified by centrifugation at 15,000 × g for 5 min at 4 °C. The protein concentration (~5
mg/ml) was determined by the Lowry method, and the nuclear extracts
were rapidly frozen and stored at Electrophoretic Mobility Shift Assay--
Calu-3 cells were
placed in medium without serum for 16-18 h, and IL-1 Plasmid Constructions--
The ( Transfections and Assays of Reporter Gene
Constructs--
Plasmids for transfection were purified with the
Qiagen endo-free plasmid Mega kit. To avoid any possible influence of
the quality of plasmid preparation on transfection efficiency, each series of transfection experiments was performed using products from at
least two different amplifications. Calu-3 and T84 cells were
transiently transfected in six-well dishes with LipofectAMINE Plus
reagent (Life Technologies) and 2 µg/well DNA in opti-MEM medium
(Life Technologies) for 20 h, according to the manufacturer's directions. The transfected cells were washed three times with cold PBS
and scraped off into 240 µl of lysis buffer (Promega). Each lysate
was mixed vigorously and clarified by centrifugation at 12,000 × g for 3 min at 4 °C. Supernatants were used for reporter assays. The luciferase activity in 20- or 30-µl extract was evaluated with the Luciferase Reporter assay system (Promega) and a Berthold Biolumat LB9500 luminometer.
Effect of IL-1
Treatment of Calu-3 cells with various doses of IL-1 Influence of IL-1 The Enhanced CFTR Promoter Activity by NF-
The reproducible 1.4- to 2-fold induction of CFTR promoter
activity after cotransfection with p50/p65 expressing vectors or after
IL-1 The development of an inflammatory response is a complex
biological process that involves many changes in gene expression in
populations of interacting cells, all with different time courses. In
the microenvironment of the inflammation site, this complex cascade of
events produces extracellular signals, such as IL-1 IL-1 Studies on the IL-1 The subtle difference between the The increased CFTR expression in response to NF- and interferon
reduce the expression of the cystic fibrosis transmembrane
conductance regulator (CFTR) gene (CFTR) in HT-29 and T84
cells by acting post-transcriptionally. We have investigated the effect
of the pro-inflammatory peptide interleukin 1
(IL-1
) on the
expression of the CFTR in Calu-3 cells. IL-1
increased
the production of CFTR mRNA in a dose- and
time-dependent manner. Its action was inhibited by
inhibitors of the NF-
B pathway, including
N-acetyl-L-cysteine, pyrrolidine
dithiocarbamate, and a synthetic cell-permeable peptide containing the
NF-
B nuclear localization signal sequence. Gel shift analysis showed
that IL-1
activated NF-
B in Calu-3 cells, and transfection
experiments using p50 and RelA expressing vectors showed that exogenous
transfected NF-
B subunits increased the concentration of CFTR
mRNA. Gel shift analysis with antibody supershifting also showed
that IL-1
caused the binding of NF-
B to a
B-like response
element at position
1103 to
1093 in the CFTR
5'-flanking region. Transfection experiments using
2150 to +52
CFTR reporter gene constructs showed that the activity of
the CFTR promoter is enhanced by exogenous transfected
NF-
B and IL-1
and that this enhancement is due, at least in part,
to the
1103 to
1093
B site. We conclude that the intracellular
signaling that leads to increased CFTR mRNA in response to IL-1
in Calu-3 cells includes the binding of NF-
B to the
1103
B
element and a subsequent increase in CFTR promoter activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and INF
,
modulate the expression of the CFTR by acting
post-transcriptionally (6, 7). However, the effects of other
pro-inflammatory cytokines, such as interleukin-1
(IL-1
), on
CFTR expression are poorly documented (8). TNF
and
IL-1
, which have overlapping and synergistic effects on cell
function, should be considered to be proximal or primary cytokines, in
many respects, because they are produced early in the response to
infection and determine the pattern of later cytokine production and
secretion in the inflammatory response. The signaling pathways mediated
by the receptors for both IL-1
and TNF
are complex and involve
multiple coordinated kinases, including JNK, SAP/p38 and ERK2 MAP
kinases. These activate transcription factors such as AP-1 and NF-IL6
(C/EBP
). The receptor-signaling pathways ultimately converge upon
the NF-
B-inducing kinase, which activates the I
B kinase complex.
Phosphorylation of the NF-
B cytoplasmic inhibitory binding protein,
I
B, by I
B kinase triggers its ubiquitination/degradation
and allows the release of the active form of the NF-
B factor. Thus,
we need to know whether IL-1
, like TNF
, modulates CFTR
expression, and whether this effect depends on transcription factors
such as NF-
B.
B is clearly involved in the inducible regulation of genes in the
immune system and the inflammatory response. NF-
B consists of
dimeric complexes of Rel/NF-
B proteins sequestered in the cytosol by
the inhibitory proteins I
B. The phosphorylation and degradation of
I
B leads to the translocation of NF-
B to the nucleus, where it
binds to specific cis-regulatory elements located in
transcriptional regulatory regions of its target genes.
48 (with respect to the initiation
site by Yoshimura et al. adjacent to an inverted CCAAT
element (Y box) at
60 (12, 13). Li et al. (14) recently
suggested that the transcription of CFTR is regulated in
part by factors directing modifications of chromatin and interacting
with the Y box element. However, other more distal regulatory regions
within the CFTR 5'-region may also be responsible for basal and protein
kinase A-mediated gene expression (13). These regions include
putative AP-1 elements (at positions
976 and
1058) close to a DNase
I-hypersensitive region specific to cells expressing the
CFTR (around
950) (10).
on the expression
of the CFTR in Calu-3 cells. Northern blot analysis showed that IL-1
stimulated the production of CFTR mRNA via
an NF-
B-dependent mechanism. Electrophoretic mobility
shift assays and reporter transfection assays indicated that a
B
element at position
1103 of the CFTR 5'-flanking region
was involved in this regulation.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and active and inactive forms
of NF-
B NS 50 peptides were purchased from Calbiochem,
N-acetyl-L-cysteine (NAC), and pyrrolidine
dithiocarbamate (PDTC) was from Sigma-Aldrich.
-actin cDNA probe from Oncogene Science (France
Biochem, Meudon, France). The mRNAs were quantified by
densitometric scanning of the autoradiograms using an ImageMaster VSD
(Amersham Pharmacia Biotech, Orsay, France)
80 °C.
(Calbiochem,
France) (2 ng/ml) was then added to the medium. Oligonucleotides,
synthesized by Eurobio (France), were annealed, end-labeled with
[
-32P]dATP (50 µCi at 3000 Ci/mmol, Amersham
Pharmacia Biotech) using the T4 polynucleotide kinase (Life
Technologies) and purified on micro-spin purification columns (Qiagen,
France). Binding reactions were done by mixing ~40 fmol of
double-stranded, end-labeled oligonucleotides with 10 µg of nuclear
extract proteins for 20 min at room temperature in a final volume of 20 µl. The mix contained EMSA binding buffer (10 mM HEPES,
pH 7.9, 10 mM KCl, 0.5 mM DTT, 0.5 mM EDTA, 5% glycerol, and 0.2-1 µg of poly(dI-dC)).
Unlabeled double-stranded competitors were added to the binding
reaction mixture 10 min prior to adding labeled probe. Supershift
analyses were done by incubating nuclear extracts with 1.5 ng of the
appropriate antibody (anti p50, RelA, or cRel from Santa Cruz
Biotechnology, Inc., Tebu, France) for 30 min on ice prior to
adding32P-labeled probe. Samples were loaded onto a 5%
nondenaturing polyacrylamide gel in 0.5× Tris borate EDTA, without any
loading dye, and electrophoresed at 125 V for 2.5 h. The separated
DNA·protein complexes were visualized on gels by autoradiography at
80 °C for 4-16 h. All experiments were done at least three times.
2150/+52)pGL3 construct was
obtained by subcloning the SacI/HindIII 2.2-kb
fragment of the (
2150/+52)-luc construct, generously provided by Dr.
G. S. McKnight (University of Washington, Seattle, WA), into the
pGL3 basic vector (Promega, Charbonnières, France). In
vitro site-directed mutagenesis was performed on this plasmid
using the Stratagene mutagenesis system following the manufacturer's
instructions. The sequences of the (
2150/+52)pGL3 constructs were
confirmed by DNA sequencing (Genome Express, France). Two independent
clones were tested in transfection experiments and gave similar
luciferase activities. CMV-driven expression plasmids for p50 and RelA
were kindly provided by Dr. Bauerle (Tularik, San Francisco, CA). A
-galactosidase-encoding vector, CMV-
-galactosidase, was purchased
from CLONTECH.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
on CFTR mRNA in Calu-3 Cells--
Two
pro-inflammatory cytokines, TNF
and INF
, have been reported to
down-regulate CFTR expression in colonic cell lines (6, 7).
We determined whether this response to pro-inflammatory mediators was a
general phenomenon by testing the ability of TNF
and IL-1
to
modulate CFTR expression in colonic (HT-29) and pulmonary (Calu-3) cell lines. Cultures of HT-29 and Calu-3 cells were stimulated for 24 h with TNF
or IL-1
, and CFTR mRNA was measured by
Northern hybridization analysis. TNF
reduced the amount of CFTR
mRNA in HT-29, as it has already been described (6, 7), but
increased it slightly in Calu-3 cells (Fig.
1A). These results contrast with those obtained with IL-1
, which significantly increased the
CFTR mRNA in both cell lines. The increase in CFTR mRNA in Calu-3 cells induced by IL-1
treatment was always greater than that
induced by TNF
, under our experimental conditions.
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Fig. 1.
Northern blot analysis of CFTR mRNA in
Calu-3 and HT-29 cells following stimulation with
IL-1 and/or TNF
.
A, HT-29 and Calu-3 cells were incubated with or without 15 ng/ml TNF
or 2.5 ng/ml IL-1
for 24 h. Total RNA was then
prepared, fractionated on agarose gel, transferred onto nylon
membranes, and hybridized with the appropriate cDNA probe (CFTR,
-actin). A Northern blot autoradiograph representative of three
independent experiments is shown. B, Calu-3 cells were
incubated for 30 min with 30 mM
N-acetyl-L-cysteine (NAC) or 100 µM pyrrolidine dithiocarbamate (PDTC) and
subsequently stimulated with increasing concentrations of IL-1
(0.5, 1.0, and 2.5 ng/ml) for 24 h (left panel), with 18 mM NF-
B-NS50 (NF-
B NLS peptide), and with
its inactive form (control peptide) for 24 h (right
panel). The total RNA were then subjected to Northern blot
analysis as in A. C, the IL-1
-induced increase
in CFTR mRNA. Left panel, a typical Northern blot
obtained after incubating the cells with IL-1
(2 ng/ml) for the
indicated time. Right panel, the mRNAs were quantified
by densitometry and the amounts of CFTR mRNA were normalized to
those of
-actin. The results are expressed as ratios of arbitrary
units.
(0.5, 1.0, and
2.5 ng/ml) increased the amount of CFTR mRNA in a
dose-dependent manner (Fig. 1B, upper
bands), whereas the mRNA of the constitutively expressed
control,
-actin, was stable under all conditions (Fig. 1B, lower bands). We investigated the time course
of this effect by incubating Calu-3 cells with IL-1
(2 ng/ml) for 0, 3, 6, 9, 15, and 24 h and analyzing the CFTR mRNA by Northern
blotting (Fig. 1C). Fig. 1C (right
panel) shows the kinetic of the CFTR mRNA increase in response
to IL-1
. IL-1
caused a rapid increase in steady-state levels of
CFTR mRNA and continued to exert its effect for up to 24 h
thereafter. Thus, these data indicated that the effect of IL-1
on
CFTR mRNA production is dose- and time-dependent.
on CFTR Gene Expression: Role of an
NF-
B-dependent Pathway--
Because IL-1
is known to
activate NF-
B in several cell types, we determined whether the
IL-1
-induced expression of CFTR mRNA was mediated by an
NF-
B-dependent mechanism. In the first series of
experiments, the cells were incubated with
N-acetyl-L-cysteine (NAC) and pyrrolidine
dithiocarbamate (PDTC), two radical scavengers that inhibit I
B
phosphorylation/degradation and, consequently, NF-
B activation. In
the second series of experiments, the cells were pretreated with a
synthetic peptide containing the NF-
B nuclear localization signal
(NLS) fused to a membrane-permeable hydrophobic region, which
specifically prevents NF-
B nuclear translocation, or with its
inactive analogue (15, 16). The cells were then stimulated with
IL-1
. NAC and PDTC did not modulate the amount of constitutive CFTR
mRNA but markedly reduced the IL-1
-mediated increase in CFTR
mRNA (Fig. 1B). Similar results were obtained with cells
pretreated with the NF-
B NLS but not those pretreated with its
inactive form. These results suggest that IL-1
acts on
CFTR expression, at least in part, via an
NF-
B-dependent pathway. We investigated the link between
IL-1
, NF-
B, and the expression of the CFTR using two
experimental strategies. We first measured the NF-
B activity by EMSA
using nuclear extracts from Calu-3 cells treated with IL-1
and a
labeled consensus
B probe (mouse immunoglobulin kappa light chain
enhancer,
B element). IL-1
activated NF-
B, as indicated by the
appearance of DNA·NF-
B complexes (Fig.
2). We also transiently transfected
Calu-3 and T84 cells (a human intestinal cell line that also expresses
the CFTR constitutively) with CMV-driven RelA (p65) and p50
expressing vectors, or the empty vector (mock control), and then
subjected them to Northern blot analysis. We checked that this
transfection procedure mimicked the endogenous activation of the
NF-
B factor by looking for specific factors binding the
B element
in the nucleus of the p50/p65-transfected cells (Fig. 2). The relative amounts of CFTR mRNA in NF-
B-expressing cells were greater than those of the control cells (Fig. 3).
Hence, IL-1
activates NF-
B in Calu-3 cells, and the generation of
active NF-
B can lead to the up-regulation of CFTR
expression.
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Fig. 2.
NF- B DNA binding
activity in Calu-3 cells. Electrophoretic mobility shift assay
using a consensus
B element as probe and nuclear extracts from
control (lanes 1-3), IL-1
-stimulated (lanes
4-8), mock transfected (lane 9), and
p50/RelA-transfected cells (lane 10). The specificity of the
labeled complex is illustrated by the ability of a 40-fold molar excess
of unlabeled probe to prevent its formation (lanes 2 and
5) and by the incapacity of the same molar excess of an
unrelated oligomer (AP-2 response element) to produce the same effect
(lanes 3 and 6). The specificity of the labeled
complexes is also shown by supershift analysis using an anti-I
B
antibody as control (lane 7) and an anti-RelA antibody
(lane 8), which prevents its formation. Finally, the nuclear
extracts from p50/RelA-transfected cells (lane 10) produced
the same shifted complex, further indicating that this complex
corresponds to NF-
B. The arrow denotes the
NF-
B-specific complex.
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Fig. 3.
Northern blot analysis of the CFTR mRNA
in T84 and Calu-3 cells expressing exogenous transfected
NF- B p50 and RelA subunits. T84 and
Calu-3 cells were transfected with 1.4 µg and 0.6 µg/well of
CMV-P50- and CMV-RelA-expressing vectors or with 2 µg of CMV empty
vector in 35-mm dishes for 24 h. Total RNA was extracted and
subjected to Northern blot analysis to detect CFTR (top) and
-actin (bottom) mRNAs. There was more CFTR mRNA
in Calu-3 cells (left) and T84 cells (right)
transfected with NF-
B-expressing vectors (p50/RelA) than
in cells transfected with the control vector (mock).
1103 to
1093 Region as a Putative NF-
B Response
Element--
Previous studies on the CFTR promoter have
shown that the CFTR 5'-flanking region is sufficient to trigger the
tissue-specific, low activity of the CFTR (9, 10). If the
NF-
B factor acts on CFTR transcription, there must be an
NF-
B response element in a region regulating the transcription of
the CFTR gene, and in particular, in the 5'-flanking region.
Analysis of the sequence of this region revealed a putative NF-
B
response element between
1103 and
1093 (5'-GGGAATGCCC-3') that
differed by 1 bp from the consensus NF-
B response element
(GGGNNTYYCC) (17). We next investigated whether the action of NF-
B
on CFTR mRNA production involved the binding of NF-
B to this
putative cis-regulatory sequence. We used an electrophoretic
mobility shift assay (EMSA) to determine the capacity of the
1103 to
1093 sequence to bind NF-
B proteins using the
1111 to
1090
region of CFTR as probe and nuclear extracts from Calu-3
cells treated with IL-1
(2 ng/ml) (Fig.
4). Cells incubated with IL-1
formed a
single shifted complex. This complex was displaced by excess unlabeled
consensus
B element from the mouse immunoglobulin kappa light chain
enhancer (B site), whereas the same excess of unlabeled consensus AP-2
element did not change the binding of the labeled probe (Fig. 4,
compare lanes 4 and 5). Thus, a specific
IL-1
-induced factor that has a high affinity for the consensus
B
element binds to the
1111 to
1090 probe. We attempted to identify
the proteins that formed the shifted complex in a supershift analysis
using antibodies to three members of the NF-
B/Rel family (p50, RelA
(p65), and cRel). The formation of the shifted complex was prevented by
anti-p50 or anti-RelA but not by anti-cRel antibodies (Fig. 4,
lanes 7-9).
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Fig. 4.
DNA binding activity of the 1111 to
1090
region. Gel retardation analysis of the
1111 to
1090 region.
32P-Labeled oligomers corresponding to the
1111 to
1090
region of the CFTR promoter were incubated with nuclear extracts from
control (lane 2) and IL-1
-stimulated (lanes
3-9) Calu-3 cells. The DNA·protein complex indicated by the
arrow was present only in IL-1
-stimulated cells.
Competition analysis of the shifted complex (lanes 4 and
5). The formation of the IL-1
-induced complex was
specifically inhibited by a 40-fold molar excess of a consensus
B
element (lane 4, oligo
B) but not
by the same excess of an unrelated oligomer corresponding to the
binding sequence for AP-2 factor (lane 5, oligo
AP2). Supershift analysis of the shifted complex (lanes
7-9): anti-p50 (lane 7,
p50), RelA (lane 8,
RelA), and cRel (lane 9,
cRel) were added to the nuclear extracts prior to
32P-labeled probe. No nuclear extracts were added in
lane 1.
B--
NF-
B (probably
the p50/RelA heterodimer) bound the
1103
B element in
vitro. Thus, this sequence could constitute a
B regulatory element in the CFTR 5'-flanking region. We investigated this
and determined whether NF-
B acted on the transcription of the
CFTR by constructing a human CFTR
promoter-luc plasmid, (
2150/+52)pGL3, and transfecting
Calu-3 cells with it (Fig.
5A). Cotransfection experiments using this construct plus the CMV-p50- and RelA-expressing vectors, or the mock plasmids as control, showed a 2.0-fold increase in
the promoter activity when the cells were cotransfected with the
CMV-p50 and RelA plasmids. Thus the CFTR promoter activity was stimulated by NF-
B subunits. We then generated a site-specific mutation of the
1103
B element in the (
2150/+52)pGL3 construct by changing two nucleotides within the
1103 to
1093 sequence (from
5'-GGGAATGCCC-3' to 5'-GGGCATTTCT-3'). This base transition was
sufficient to destroy the binding capacity of the NF-
B proteins (data not shown). The activity of mutated CFTR promoter was
less strongly stimulated (1.4-fold induction) than was the wild type one when this reporter construct was cotransfected with p50- and RelA-expressing vectors (Fig. 5A). Hence, the
1103 to
1093
B element of the CFTR 5'-flanking region may take
part in the up-regulation of the CFTR by exogenous
transfected NF-
B factor. Finally, we performed transfection
experiments with the (
2150/+52)pGL3 reporter gene constructs and
stimulated cells with IL-1
to determine whether NF-
B activation
mediated by IL-1
involved this
B element. IL-1
produced more
luciferase activity in cells transfected with the wild type constructs
than in cells transfected with the mutated one (Fig. 5B).
These results indicated that IL-1
stimulation reproduced the results
obtained with exogenous NF-
B factor
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Fig. 5.
Activities of control and mutated
CFTR promoter in Calu-3 cells. A,
Calu-3 cells were transiently transfected using 1.4 µg of
( 2150/+52)pGL3 or mutated (
2150/+52)pGL3, or the pGL3 basic vector
(control) in combination with 0.6 µg/well CMV-driven expression
vectors for p50 (0.2 µg/well) and for RelA (0.4 µg/well), or with
0.6 µg/well of the empty CMV vector alone to keep the concentration
of transfected DNA constant. The results are expressed as the ratio of
the luciferase activities in the (
2150/+52)pGL3 constructs to the
activities in the control constructs. Promoter activity is given as the
mean of triplicate assays for one of two experiments. The parentheses
show the -fold increase in luciferase activity in cells transfected
with p50/RelA NF-
B plasmids compared with the luciferase activity in
the cells transfected with the reporter constructs without p50- and
RelA-expressing vectors. B, Calu-3 cells were transiently
transfected as previously with the same pGL3 vectors in combination
with a CMV-
gal vector to normalize for transfection efficiency.
36-h post-transfection cells were stimulated with IL-1
for 16 h
in a medium deprived of serum. Promoter activity is expressed as
the ratio of the luciferase/
-galactosidase activities in the
(
2150/+52)pGL3 constructs to the activities in the control
constructs. Data are expressed as the means ± S.E. of three
independent experiments, each in triplicate. **, p < 0.005, using Student's t test for unpaired values.
treatment is within the range of induction reported for the
same promoter after stimulation with forskolin (12, 13). The modest
increase caused by Il-1
was abolished when the
1103
B element
was mutated. Thus, the transfection data support the Northern blot
analyses presented in Figs. 2 and 3. Therefore, we believe that this
inducibility of the CFTR promoter could be biologically
important and conclude that the
1103
B element of the
CFTR takes part in the induction of CFTR mRNA production in response to IL-1
and in the subsequent NF-
B activation in Calu-3 cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and TNF
, that
tightly control the changes in gene expression. This response helps to
restore the equilibrium disturbed by the initial injury (bacterial,
viral, or parasitic infection), but excessive production of
inflammatory mediators may have negative effects and can lead to the
destruction of the damaged tissue. Such a failure to modulate the
inflammatory response occurs in cystic fibrosis. The abundance of
pro-inflammatory cytokines in the airways of CF patients reflects the
dramatic lung inflammatory injury that is often lethal in cystic
fibrosis. Several studies have focused on the effect of cytokines in
the inflamed CF airways on the expression of the CFTR (6,
7). These studies have shown that CFTR expression is
modulated by inflammatory signals, suggesting that CFTR contributes to
the change in cell functions caused by the inflammatory stress. We have
now obtained evidence that IL-1
stimulates the production of CFTR
mRNA in a dose-, time-, and NF-
B-dependent manner in
a pulmonary cell line derived from the serous cells of submucosal
glands. The EMSA and promoter-reporter gene transfection assays
indicate that IL-1
causes NF-
B to bind to the CFTR
5'-flanking region, leading to increased CFTR transcription. This is of particular interest, because molecules, such as forskolin, that increase intracellular cAMP were the only extracellular stimuli that increased CFTR transcription until now. The cells were
incubated with IL-1
for close to 20 h in most experiments, so
that the observed effect on CFTR expression reflects primary
and possibly secondary Il-1
-induced transduction pathways.
and TNF
have broadly overlapping, often synergistic, effects
on cell functions. Not surprisingly, these cytokines also share
signaling pathways, such as those that activate the NF-
B transcription factor and the p38, JNK, and ERK MAP kinases. Our data
for Calu-3 cells show that TNF
and IL-1
stimulate the expression of CFTR mRNA but to different extents. Surprisingly, the two
cytokines have opposing effects in HT-29 cells, TNF
decreases CFTR
mRNA expression, whereas IL-1
increases it. This finding
suggests that IL-1
involves signaling events that are not shared
with TNF
. One possible explanation is that TNF
and IL-1
signaling pathways stimulate different signals and/or activate the same signal but confer on it different properties under some conditions.
signaling pathway have often focused on the
complex cascade of intracellular molecular events that converge ultimately on the activation of other transcription factors, including AP-1 (18) and C/EBP (19). However, the IL-1 receptor-mediated signaling
pathway also activates the NF-
B transcription factor (20, 21), which
is of central importance to immune and inflammatory responses. Our
findings provide evidence that the IL-1
-induced expression of the
CFTR is mediated by NF-
B activation and binding to the
CFTR 5'-flanking region. Nonetheless, activation of NF-
B does not appear to be sufficient, by itself, to activate
CFTR transcription, as indicated by the effects of TNF
in
HT-29 and Calu-3 cells. Also, TNF
is a strong activator of NF-
B
in both cell lines (data not shown), it inhibits CFTR mRNA
expression in HT-29 cells and only slightly stimulates CFTR
expression in Calu-3 cells. These findings suggest that regulation of
CFTR transcription by IL-1
, which we unmasked in Calu-3
cells, involves different IL-1
-induced transcription factors in
addition to NF-
B. This is supported by the presence of several
putative binding sequences for IL-1-induced transcription factors in
the CFTR 5'-flanking region, including two AP-1 response
elements in the vicinity of the
1103
B element (13). There is
strong evidence for interactions between NF-
B and bZIP transcription
factors, such AP-1 and C/EBP, influencing the ability of NF-
B to
regulate gene expression in a selective manner (22-26). Thus,
interactions between these factors could lead to a synergistic
activation of the CFTR transcription.
1103
B element and the consensus
B motif probably explains the slight difference in NF-
B binding
affinity, between the
1103
B element and the canonical
B
element from the immunoglobulin kappa light chain enhancer (in
vitro assay, data not shown). Nevertheless such variant NF-
B binding sites have been found to be important in gene transcription (26), because protein·protein interactions with other transcription factors are believed to stabilize their binding to DNA. Thus, interactions between NF-
B and other IL-1
-induced transcription factors could stabilize NF-
B binding to the CFTR
5'-flanking region.
B
activation could be of pathophysiological significance in cystic
fibrosis, because it was recently demonstrated that NF-
B is
endogenously activated in CF cells. The endogenous activation of
transcription factor NF-
B has been correlated with the constitutive
inflammatory response in CF cells (27). It is suggested that the
presence of the F508del-mutated CFTR proteins in the cells
produces an endogenous stress that results in increased NF-
B
activation and thus an increased production of endogenous
pro-inflammatory cytokines. The present study shows that the activation
of NF-
B can also lead to increased CFTR activity in
airway epithelial cells. Thus, the F508del protein could exert a
positive influence on its own synthesis in some conditions by
activating the NF-
B system.
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ACKNOWLEDGEMENTS |
---|
We thank Marc Lombes (INSERM U. 478) for help in initiating this study, Michel Raymondjean (INSERM U. 129) for helpful discussion, and Pascale Fanen and Bruno Coste (INSERM U. 468) for help in DNA sequencing. We also thank Dr. G. S. McKnight (University of Washington, Seattle, WA) for CFTR promoter-containing plasmids and Dr. Bauerle (Tularik, San Francisco, CA) for expression vectors for p50 and RelA. The English text was edited by Owen Parkes.
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FOOTNOTES |
---|
* This work was supported in part by the INSERM, CNRS, Association Française de Lutte contre la Mucoviscidose.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.
§ Supported by a fellowship from the Association Française de Lutte contre la Mucoviscidose and Fondation pour la Recherche Médicale.
To whom correspondence should be addressed: Tel.:
33-1-40-61-56-21; Fax: 33-1-40-51-55-91; E-mail:
edelman@necker.fr.
Published, JBC Papers in Press, December 12, 2000, DOI 10.1074/jbc.M006636200
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ABBREVIATIONS |
---|
The abbreviations used are:
CF, cystic fibrosis;
CFTR, cystic fibrosis transmembrane conductance regulator;
TNF, tumor necrosis factor
;
IFN
, interferon
;
JNK, c-Jun
NH2-terminal kinase;
SAP, stress-activated protein;
ERK, MAP, mitogen-activate protein;
EMSA, electrophoretic mobility shift
assay;
NAC, N-acetyl-L-cysteine;
PDTC, pyrrolidine dithiocarbamate;
kb, kilobase(s);
PBS, phosphate-buffered
saline;
DTT, dithiothreitol;
CMV, cytomegalovirus;
NLS, nuclear
localization signal;
bp, base pair(s).
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