From the Instituto de Investigaciones
Bioquímicas-Fundación Campomar (IIB, UBA; IIBBA, CONICET)
and the § Centro Nacional de Genética Médica,
(ANLIS), 1405 Buenos Aires, Argentina
Received for publication, November 6, 2000, and in revised form, January 29, 2001
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ABSTRACT |
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Interleukin-1 beta (IL-1 Cystic fibrosis (CF)1 is
an inherited disorder associated with severe inflammation, repeated
infection, and colonization by Pseudomonas aeruginosa and
Staphylococcus aureus (1). Before lung infection, an
excessive release of proinflammatory cytokines and an increased number
of neutrophils has been reported in the bronchoalveolar lavage fluids
of CF patients (2), which also contain high levels of mucus.
We have recently reported that the proinflammatory cytokine,
interleukin-1 beta (IL-1 There are some reagents that can stimulate or inhibit the activity of
NF- Cell Culture and Treatments--
T84 human colon carcinoma cells
(American Type Culture Collection (ATCC) cell line CCL248; Manassas,
VA) were grown in Dulbecco's modified Eagle's medium F-12 1:1 mixture
supplemented with 10 units/ml penicillin, 10 mg/ml streptomycin, and
5% fetal bovine serum (Life Technology, Inc.). Subconfluent cells
(70-75%) were incubated in serum-free medium (10 ml/100-mm Petri
dish) for 48 h. Then IL-1 T84 Transduction--
The T84 cell line was cultured to
subconfluency and transduced with Ad5I Preparation of Total RNA--
AD5I Northern Blot Analysis--
Northern blot analysis was performed
as previously described (10). Briefly, equal amounts of total RNA (30 µg) were separated electrophoretically on 1% agarose gels containing
2.2 M formaldehyde and transferred to Zeta Probe GT
blotting membranes (Bio-Rad). RNA was stained with a solution of 0.04%
methylene blue in 0.5 M sodium acetate (pH 5.2),
scanned, and quantified (NIH Image program)2 to control for
sample loading. The membranes were hybridized at 65 °C with a
3.3-kbp CFTR cDNA probe (ATCC 61136) and with a 1.25-kbp
glyceraldehyde-3-phosphate dehydrogenase cDNA probe, both labeled
with [ Nuclear Extract Preparation and Electrophoretic Mobility Shift
Assay (EMSA) Analysis (Gel Shift)--
After stimulation, the cells
were washed twice with 1 ml cold phosphate-buffered saline, scraped
into 1 ml cold phosphate-buffered saline, and centrifuged at 4 °C
for 20 s at 14,000 × g in a microcentrifuge. The
cell pellet was resuspended in 500 µl of buffer containing 10 mM Hepes (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM fresh dithiothreitol, and
protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, and 30 µg/ml leupeptin). The cell pellet
suspension was lysed with a Dounce homogenizer, and the extent of lysis
was determined by trypan blue exclusion. The cell lysate/nuclear
suspension was prepared by centrifugation (14,000 × g
for 2 min at 4 °C in a microcentrifuge). The pellet was resuspended
in 50 µl of buffer (20 mM Hepes (pH 7.9), 25% glycerol,
0.42 M NaCl, 1.5 mM MgCl2, 0.2 EDTA, 0.5 mM dithiothreitol, and protease inhibitors) and left on ice for 1 h with occasional vortexing. Extracted nuclei were isolated by further centrifugation at 14,000 × g
for 5 min at 4 °C (11). Protein concentration was determined using
the method of Bradford (Bio-Rad). Oligonucleotide probes containing the
consensus sequence for NF- Western Blot Analysis--
Subconfluent monolayers of T84 cells
(grown for 2 days in serum-free Dulbecco's modified Eagle's medium
F-12) were incubated for 1 h in the presence or absence of IL- Quantification and Statistical Analysis--
Northern and
Western blots were scanned on an HP4C scanner and quantified using the
PC-compatible program, NIH Image.2 Sample loading in
Northern blots was quantified with methylene blue staining. A lineal
response to methylene blue staining was obtained for up to 40 µg of
total RNA (3). Therefore, a maximum of 30 µg was used in each assay.
Statistical analysis was performed using analysis of variance (ANOVA)
and the Tukey test.
To determine whether the CFTR response to IL-1) regulates the levels
of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA
and protein in the T84 human carcinoma cell line. Here, we studied the
role of the transcription factor NF-
B in this regulation. Initially, T84 cells were pretreated with the NF-
B inhibitor pyrrolidine dithiocarbamate. Cells were then stimulated with IL-1
, and
CFTR mRNA levels were determined after 4 h by Northern blot
analysis. As a result of PDTC treatment, IL-1
stimulation of CFTR
mRNA was blocked. On the other hand, daunorubicin, an NF-
B
activator, increased the steady-state levels of CFTR mRNA.
Furthermore, after treatment with IL-1
for 1 h, cytoplasmic
I
B
degradation occurred simultaneously with translocation of p65
into the nucleus. The T84 cells were also transduced with an adenoviral
vector expressing a dominant negative form of I
B
, which prevents
I
B
phosphorylation and the subsequent nuclear translocation of
NF-
B. After viral transduction, the cells were stimulated with
IL-1
for 4 h, and CFTR mRNA levels were measured by
Northern blot analysis. The stimulation of CFTR, induced by IL-1
,
was also blocked in the presence of the dominant negative mutant. These
results indicate that NF-
B is involved in the pathway by which
IL-1
regulates CFTR.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
), regulates the steady-state levels of CFTR
mRNA and protein in the T84 human carcinoma cell line in a biphasic
way: stimulatory at 0.5 ng/ml and inhibitory at higher doses (3).
However, the mechanism involved in this response of CFTR to IL-1
is
essentially unknown. In most cells, IL-1
causes translocation of the
transcription factor NF-
B into the nucleus (4). This transcription
factor is a heterodimer (composed most commonly of p65/p50), the
constituents of which are members of the Rel family of transcription
factors (5). NF-
B resides in the cytoplasm as an inactive complex,
bound to the endogenous cytoplasmic inhibitors known as I
Bs. The
best characterized and most extensively studied NF-
B inhibitor is
I
B
. In response to extracellular stimuli, such as IL-1
, the
I
B
protein is rapidly phosphorylated by the I
B kinase complex
and targeted for proteolysis (6). This exposes the nuclear localization
sequence of NF-
B and facilitates its translocation into the nucleus
with subsequent initiation of gene transcription (4, 5). It has been
established that I
B kinase phosphorylates I
B
on serine
residues 32 and 36 and that this phosphorylation is the prerequisite
for ubiquitination and proteosome-dependent degradation of
I
B
. This, in turn, causes the liberation of NF-
B and allows it
to translocate into the nucleus.
B such as daunorubicin (7) and pyrrolidine dithiocarbamate (PDTC)
(8), respectively. Furthermore, Jobin and colleagues (9) have
constructed an adenoviral vector bearing a mutant form of I
B
in
which serines 32 and 36 are replaced by alanine residues (S32A/S36A)
thereby preventing I
B
phosphorylation and degradation. The
I
B
mutant has been shown to act as a very specific NF-
B
superrepressor (9). Using these reagents, we have demonstrated here
that NF-
B is involved in the up-regulation of CFTR by IL-1
in T84 cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
was added to the medium at different
concentrations as indicated in the legends to the figures. For NF-
B
inhibition experiments, the cells were preincubated with PDTC (100 µM) for 30 min before the addition of IL-1
. For
NF-
B stimulation experiments, the cells were incubated with
daunorubicin (0.1 µM) for 1 h for gel shift assays
and for 4 h for Northern blots.
B
or Ad5LacZ in serum-free
medium (Dulbecco's modified Eagle's medium F-12; Life Technologies,
Inc.) at a multiplicity of infection of 100 (1:100 T84 cells/viral
particles) for 12 h with gentle agitation every hour. The
adenovirus was washed off, fresh serum-containing medium was added, and
the cells were incubated for a further 12 h. The cells were
incubated in serum-free medium for 48 h and then stimulated
with IL-1
(0.5 ng/ml) for 4 h.
B
- or Ad5LacZ-transduced
or non-transduced cells or treated with PDTC or daunorubicin were
stimulated with IL-1
(0.5 ng/ml) for 4 h. Total RNA was
isolated using Trizol reagent following the manufacturer's
instructions (Life Technologies, Inc.). The RNA was precipitated with
50% isopropanol, and the
A260/A230 and A260/A280 ratios were
determined to verify RNA purity.
-32P]dCTP (3000 Ci/mmol; PerkinElmer Life
Sciences) by random priming (Prime a Gene Labeling System;
Promega Co., Madison, WI), washed at 65 °C as described (10), and
exposed for various times at
70 °C with intensifying screens.
B (5'
AGT TGA GGG GAC TTT CCC AGG C
3')
were purchased from Promega (Promega Co.). Nuclear extracts (10 µg) were incubated with 5000 cpm of 32P-end-labeled
oligonucleotide probe, 0.05 mg/ml of poly(dI-dC)·poly(dI-dC), 10 mM Tris (pH 7.5), 50 mM NaCl, 5% glycerol, 1 mM EDTA (pH 8.0), and 0.2 mg/ml bovine serum albumin, in a
final volume of 10 µl for 20 min at room temperature. The complexes
were fractionated on 4% native polyacrylamide gels, run in 2.2 M formaldehyde-TBE buffer at 4 °C. Gels were dried and
exposed to Kodak film at
70 °C.
at two concentrations (0.5 or 2.5 ng/ml). The monolayers were washed
with ice-cold phosphate-buffered saline, and lysed in radioimmune
precipitation buffer (25 mM Tris, 150 mM NaCl,
0.5% deoxycholic acid sodium salt, 1% Nonidet-P40, 2 mM
EDTA (pH 8)). Nuclei and unbroken cells were removed by centrifugation (15,000 × g for 15 min, at 4 °C). Soluble proteins
in the supernatant were denatured with Laemmli sample buffer,
fractionated by 10% polyacrylamide gel electrophoresis, and
transferred to a nitrocellulose membrane. Transferred proteins were
probed with the monoclonal anti-NF-
B antibody (Ab p65) or
anti-I
B
antibody (Santa Cruz Biotechnology, Santa Cruz, CA). As a
control for loading, histone H1 and
-actin were probed with
appropriate antibodies (Santa Cruz Biotechnology). The primary antibody
was visualized with horseradish peroxidase-conjugated anti-mouse
immunoglobulin and the enhanced chemiluminescence (ECL) Western
blotting kit (Amersham Pharmacia Biotech).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
in T84 cells
involves NF-
B activation, we first studied the DNA-binding activity of NF-
B by EMSA using as probe a double-stranded oligonucleotide possessing an NF-
B-binding site. Cells were treated with IL-1
for
15, 30, and 60 min at 2 concentrations, 0.5 ng/ml (to stimulate CFTR
expression) and 2.5 ng/ml (to inhibit CFTR expression). The nuclear
extracts were then assayed by gel shift. As shown in Fig. 1, IL-1
induced an increase in NF-
B
DNA-binding activity at 0.5 ng/ml with a maximum after 60 min. This
activation was observed as early as 15 min after IL-1
treatment. At
2.5 ng/ml IL-1
, the levels of NF-
B diminished with respect to
those induced at 0.5 ng/ml. This is in agreement with the biphasic
curve previously obtained for CFTR mRNA and protein levels in which
IL-1
is stimulatory at 0.5 ng/ml and inhibitory at higher
concentrations (3).
View larger version (35K):
[in a new window]
Fig. 1.
Interleukin-1
induces NF-
B nuclear factor in T84
cells. A, DNA binding of NF-
B in T84 cells is
stimulated with IL-1
. T84 cells were incubated for 0, 15, and 60 min
in the presence or absence of IL-1
(increasing concentrations: 0, 0.5, and 2.5 ng/ml). An NF-
B shift was induced by IL-1
treatment
(at 0.5 ng/ml) in T84 cells. Competition analysis was performed with a
100-fold excess of unlabeled probe (NF-
B). B, supershift
analysis was performed by adding monoclonal antibodies against NF-
B
p65 subunit (Ab p65), p50 subunit (Ab p50), and
p52 subunit (Ab p52). N.E., nuclear extract;
probe, 32P-labeled synthetic oligonucleotide
representing the consensus NF-
B-binding sequence. Nuclear extracts
were prepared following stimulation and analyzed for NF-
B-binding
activity as described under "Experimental Procedures."
NF-
B·DNA complexes and supershifts are shown indicating NF-
B
activation. Results are representative of three separate experiments.
C, Western blot of nuclear p65 and cytoplasmic I
B in T84
cells treated with IL-1
. T84 cells were incubated for 1 h in
the presence or absence of IL-1
. Nuclear (20 µg) and cytoplasmic
(40 µg) extracts were prepared from T84 cells treated with IL-1
(0.5 ng/ml or 2.5 ng/ml) and subjected to immunoblotting with anti-p65
or anti-I
B
antibodies. The molecular mass of I
B
is
37 kDa. Anti-
-actin and anti-histone H1 antibodies were used to
control for sample loading. Results are representative of three
separate experiments.
The NF-B complex activated by IL-1
treatment (0.5 ng/ml) of T84
cells contained a p65-p50 heterodimer as determined by supershifts using specific antibodies (Fig. 1B). This result is in
agreement with the observation that, in most cells, the heterodimer is
composed of the RelA (p65) and NF-
B1 (p50) subunits, this variant
being the most potent gene transactivator among the NF-
B family (6). Furthermore, increased levels of p65 in the nucleus were observed by
Western blotting analysis of nuclear extracts (Fig. 1C) when cells were treated with 0.5 ng/ml of IL-1
, and decreased levels were
observed at 2.5 ng/ml. These data suggest a correlation between nuclear
NF-
B levels and the CFTR response to different concentrations of
IL-1
, which might explain the biphasic curve obtained in response to
IL-1
(3).
NF-B activation by most inducers requires degradation of the isoform
I
B
. Therefore, we also looked for IL-1
-induced degradation of
I
B
in the cytoplasmic extracts of cells treated with IL-1
. Western blot analysis, using an antibody against I
B
, indicated that IL-1
effectively reduced the cytoplasmic levels of I
B
at
0.5 ng/ml, and basal levels where restored at 2.5 ng/ml (Fig. 1C). This again is in agreement with the concept that
NF-
B is no longer translocated into the nucleus when a high
concentration of IL-1
is used. This result is not surprising
because, in most mammalian cells, I
B
is rapidly degraded
following inducible phosphorylation but is quickly resynthesized in an
NF-
B-dependent manner (6). Together, these results
demonstrate that the NF-
B pathway is active in T84 cells and that
IL-1
induces the activation of NF-
B and its translocation into
the nucleus.
We next studied whether the activation and translocation of NF-B
induced by IL-1
(at 0.5 ng/ml) is involved in the regulation of CFTR
mRNA levels. The T84 cells were pretreated for 30 min with the
NF-
B inhibitor PDTC (100 µM) (12, 13). The cells were
then stimulated with IL-1
(0.5 ng/ml), and CFTR mRNA levels were
determined after 4 h, by Northern blot analysis. The inhibitor PDTC abrogated up-regulation by IL-1
but also reduced the basal CFTR
mRNA levels in a short time. However, this reduction in basal levels is compatible with the half-life (4 h) of the CFTR mRNA (3),
and a general toxic effect of PDTC does not seems to occur at 100 µM because the expression levels of
glyceraldehyde-3-phosphate dehydrogenase did not change in the presence
or absence of PDTC (Fig. 2). The results
obtained with the inhibitor PDTC, therefore, provided initial evidence
that NF-
B might be involved in CFTR regulation by IL-1
.
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To further test the proposition that NF-B activation was involved in
the increased expression of CFTR mRNA observed after IL-1
treatment at 0.5 ng/ml, daunorubicin, an NF-
B activator (7, 13), was
then used. To demonstrate that daunorubicin was effectively working in
T84 cells, an EMSA was performed using NF-
B as a probe. As expected,
an NF-
B shift was induced by daunorubicin (0.1 µM) in
T84 cells (Fig. 3B) indicating
that the dose and time-frame for the treatment was correct. Once we had
demonstrated that an effective nuclear translocation of NF-
B is
induced by daunorubicin, we examined whether CFTR mRNA levels were
also affected. Daunorubicin at 0.1 µM induces the
expression of CFTR mRNA as shown by Northern blot analysis (Fig.
3A). These results further support the notion that IL-1
,
at low doses, regulates CFTR via NF-
B activation. However, chemical
inhibitors and stimulators are not always completely specific.
Therefore, to address this possible difficulty and to confirm the role
of NF-
B T84 cells were transduced with an adenoviral vector
expressing a dominant negative form of I
B
, which prevents I
B
phosphorylation and subsequent NF-
B nuclear translocation (9). A schematic representation of the I
B
S32A/S36A construct used by Jobin and colleagues (9) to generate the adenovirus vector, is
shown in Fig. 4C. To determine
the efficiency of transduction, T84 cells were transduced with Ad5LacZ
and stained for
-galactosidase activity. An efficiency of 60-70%
was achieved (Fig. 4B). After transduction with Ad5I
B
,
the cells were stimulated with IL-1
for 4 h, and CFTR mRNA
levels were determined by Northern analysis. The stimulation of CFTR
induced by IL-1
was blocked by the dominant negative mutant, as
shown in Fig. 4A, confirming that NF-
B has a role in the
pathway by which IL-1
regulates CFTR and can stimulate the
expression of CFTR.
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The CFTR protein is not totally absent from the cell surface in cystic
fibrosis. In most cases, only a diminished amount of the protein is
normally present on the surface of CF cells (14). Therefore, any drug
that can up-regulate the gene might also facilitate an increase in the
amount of protein reaching the cell membrane, perhaps to levels
sufficient to restore a quasi-normal phenotype. It is for this reason
that it is important to identify the pathway(s) involved in CFTR
regulation. Here, we have demonstrated that IL-1 acts in the
modulation of CFTR, at least in part, through a pathway involving
NF-
B. Because IL-1
is the first recognized extracellular factor
that can increase the expression of CFTR mRNA and protein, clarification of the key role of NF-
B in the pathway of CFTR up-regulation might allow the identification of new potential targets
for therapy.
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ACKNOWLEDGEMENTS |
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The IB S32A/S36A mutant was a gift from
Dr. B. Sartor, University of North Carolina, Chapel Hill, NC.
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FOOTNOTES |
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* This work was supported with grants from the Asociación de Lucha Contra la Fibrosis Quistica (FIPAN) (to O. H. P. and T. A. S. C.), the National Research Council of Argentina (Consejo Nacional de Investigaciones Científicas y Técnicas , to T. A. S. C.), the University of Buenos Aires (to T. A. S. C.), and The Third World Academy of Sciences (to T. A. S. C.).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.
¶ To whom correspondence should be addressed: Instituto de Investigaciones Bioquímicas-Fundación Campomar, 435 Patricias Argentinas, 1405 Buenos Aires, Argentina. Tel.: 54-114-8634015; Fax: 54-114-8652246; E-mail: tasc@iib.uba.ar.
Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M010061200
2 Contact corresponding author for Web address.
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ABBREVIATIONS |
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The abbreviations used are:
CF, cystic fibrosis;
IL-1 , interleukin 1 beta;
I
B, NF-
B inhibitor;
CFTR, cystic
fibrosis transmembrane conductance regulator;
PDTC, pyrrolidine
dithiocarbamate;
EMSA, electrophoretic mobility shift assay.
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