Regulation of RANTES promoter activation in alveolar
epithelial cells after cytokine stimulation
Antonella
Casola1,
Allyne
Henderson1,
Tianshuang
Liu1,
Roberto P.
Garofalo1,2, and
Allan R.
Brasier3,4
Departments of 1 Pediatrics,
2 Microbiology and Immunology, and
3 Internal Medicine and 4 Sealy Center
for Molecular Sciences, University of Texas Medical Branch, Galveston,
Texas 77555
 |
ABSTRACT |
Regulated on activation, normal T cell
expressed, and presumably secreted (RANTES) is a member of the CC
chemokine family of proteins implicated in a variety of diseases
characterized by lung eosinophilia and inflammation, strongly produced
by stimulated airway epithelial cells. Because such cytokines as tumor
necrosis factor (TNF)-
and interferon-
(IFN-
) have been shown
to enhance RANTES induction in airway epithelial cells and RANTES gene
expression appears to be differentially regulated depending on the cell
type and the stimulus applied, in this study we have elucidated
mechanisms that operate to control RANTES induction on exposure to
TNF-
and/or IFN-
. Our results indicate that TNF-
and IFN-
synergistically induce RANTES protein secretion and mRNA expression.
RANTES transcription is activated only after stimulation with TNF-
,
but not IFN-
, which affects RANTES mRNA stabilization. Promoter
deletion and mutagenesis experiments indicate that the nuclear factor
(NF)-
B site is the most important cis-regulatory element
controlling TNF-induced RANTES transcription, although NF-interleukin-6
binding site, cAMP responsive element (CRE), and interferon-stimulated responsive element (ISRE) also play a significant role. TNF-
stimulation induces nuclear translocation of interferon regulatory factor (IRF)-3, which in viral infection binds the RANTES ISRE and is
necessary for activation of RANTES transcription. However, TNF-induced
IRF-3 translocation does not result in IRF-3 binding to the RANTES
ISRE. Although viral infection can activate an ISRE-driven promoter,
TNF cannot, indicating that RANTES gene enhancers are controlled in a
stimulus-specific fashion. Identification of molecular mechanisms
involved in RANTES gene expression is fundamental for developing
strategies to modulate lung inflammatory responses.
tumor necrosis factor; interferon; nuclear factor-
B; regulated
on activation, normal T cell expressed, and presumably secreted
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INTRODUCTION |
REGULATED ON
ACTIVATION, normal T cell expressed, and presumably secreted
(RANTES) is a member of the CC chemokine family of proteins, strongly
chemoattractant for eosinophils, basophils, monocytes, and memory T
lymphocytes (1, 34). RANTES production has been implicated
in a variety of diseases characterized by lung eosinophilia and
inflammation, including asthma and respiratory syncytial virus (RSV)
infection (2, 29, 32). Administration of RANTES in vitro
produces transendothelial migration and degranulation of eosinophils
(10) and in vivo eosinophilic inflammation in human
upper airways (3). Concentration of RANTES is
elevated in the bronchoalveolar lavage fluid of asthmatic patients
(2, 38) and children with RSV bronchiolitis (11, 15,
35). Administration of a RANTES receptor antagonist or
neutralizing RANTES antibodies completely blocks lung eosinophilia and
inflammation in experimental models of asthma (14). Airway
epithelial cells are major sites of RANTES production in these models
of lung inflammation. Cytokines produced during acute inflammation can
induce RANTES expression in different cell types. For example, tumor
necrosis factor (TNF)-
and interleukin (IL)-1 increase RANTES
secretion in fibroblast and epithelial cells, and interferon (IFN)-
has been shown to induce RANTES synergistically with TNF in
epithelial and endothelial cells (24, 36).
Human RANTES gene expression appears to be differentially
regulated depending on the cell type and the stimulus applied
(17, 25, 26, 28, 30). Induction of transcription is an
important level of control of RANTES gene expression, and different
combinations of cis-regulatory elements of the promoter seem
to be required for optimal levels of transcription in a variety of cell
types, such as monocytes, lymphocytes, and astrocytes, after
stimulation with cytokines or phorbol esters (17, 25, 26, 28,
30). In T lymphocytes, important regulatory elements of RANTES
promoter activity are the nuclear factor (NF)-
B, NF-IL-6, NF-AT, and
CD28RE binding sites (27). We and others recently showed
the essential role played by the ISRE site in virus-induced RANTES
transcription through activation and binding of transcription factors
belonging to the interferon regulatory factor (IRF) family,
specifically IRF-1, IRF-3, and IRF-7 (7, 13, 23). However,
a complete analysis of the promoter cis-regulatory elements
and NF involved in regulation of RANTES gene transcription after
cytokine stimulation of airway epithelial cells has not been performed.
The goal of this study was to define the effect of TNF-
and IFN-
stimulation on RANTES gene transcription in airway epithelial cells.
Our results indicate that TNF-
and IFN-
synergistically induce
RANTES protein secretion and mRNA expression. However, RANTES
transcription is activated after stimulation with TNF-
, but not
IFN-
, which affects RANTES mRNA stabilization. Promoter deletion and
mutagenesis experiments indicate that the NF-
B site is the most
important cis-regulatory element controlling TNF-induced
RANTES transcription, although the NF-IL-6 binding site, the cAMP
responsive element (CRE), and the interferon-stimulated responsive
element (ISRE) also play an important role. Similar to viral infection,
TNF stimulation induces nuclear translocation of IRF-3. However,
TNF-induced translocation of IRF-3 does not result in IRF-3 binding to
the RANTES ISRE. Furthermore, although viral infection can activate an
ISRE-driven promoter, TNF cannot, indicating that RANTES
gene enhancers are controlled in a stimulus-specific fashion, similar
to what we have shown for other chemokines (6).
Identification of the molecular mechanisms involved in RANTES gene
expression is fundamental for developing strategies to modulate lung
inflammatory responses.
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METHODS |
Cell culture.
A549 cells (American Type Culture Collection, Manassas, VA), human
alveolar type II-like epithelial cells, were maintained in Ham's F-12K
medium containing 10% (vol/vol) fetal bovine serum, 10 mM glutamine,
100 IU/ml penicillin, and 100 µg/ml streptomycin.
RANTES ELISA.
Immunoreactive RANTES was quantitated by a double-antibody ELISA kit
(DuoSet, R & D Systems, Minneapolis, MN) following the manufacturer's protocol.
Northern blot.
Total RNA was extracted from control and infected A549 cells by the
acid guanidinium thiocyanate-phenol-chloroform method (33). RNA (20 µg) was fractionated on a 1.2%
agarose-formaldehyde gel, transferred to a nylon membrane, and
hybridized to a radiolabeled RANTES cDNA, as previously described
(5). After the membrane was washed, it was exposed for
autoradiography using Kodak XAR film at
70°C using intensifying
screens. The membrane was stripped and reprobed for
-actin as
internal control for equal loading of the samples.
mRNA half-life measurements.
A549 cells were stimulated with TNF-
(100 ng/ml), alone or in
combination with IFN-
(100 IU/ml) for 3 h; then 100 µM
5,6-dichloro-1-
-D-ribofuranosylbenzimidazole (DRB), a
polymerase II inhibitor (9), was added to the treated cells without a change of the medium. Total RNA was isolated at time 0 of DRB addition and at multiple times thereafter. RNA
was fractionated on a 1.2% agarose-formaldehyde gel, transferred to a
nylon membrane, and hybridized to a radiolabeled RANTES cDNA. The
membrane was exposed to a PhosphorImager screen for 24 h, and
images were visualized and quantitated using PhosphorImager SI
analysis and ImageQuant software (Molecular Dynamics).
Plasmid construction and cell transfection.
5'-Deletion constructs of the human RANTES promoter were produced by
PCR using as template the full-length human RANTES promoter from
nucleotides
974 to +55 relative to the mRNA start site designated +1,
cloned into the pGL2 vector (pGL2-974), as previously described (7). Upstream primers incorporating a unique
KpnI restriction site were designed to produce
5'-deletions at nucleotides
220, 195, 150, and 120; the
downstream oligonucleotide, containing a unique HindIII
restriction site, was designed to hybridize from nucleotides +30
to +55. The PCR products were restricted with KpnI and
HindIII, gel purified, cloned into the luciferase
reporter gene vector pGL2 (Promega, Madison, WI), and named pGL2-220,
pGL2-195, pGL2-150, and pGL2-120, as previously described
(7).
Site-directed mutations of the RANTES promoter were introduced by the
PCR overlap extension mutagenesis technique using pGL2-220 as template
and mutagenic primers identical to the oligonucleotides used in
electrophoretic mobility shift assay (EMSA; Table
1), with the exclusion of the 5'-GATC
sequence, as previously described (7). The RANTES ISRE
multimer was constructed by ligation of three copies of the ISRE site
upstream of the nucleotide
54 of the human IL-8 luciferase
promoter, as previously described (6). Before
ligation, 100 pmol of the duplex oligonucleotides were phosphorylated
with T4 kinase and ligated with T4 DNA ligase, and trimers were isolated by nondenaturing gel electrophoresis (PAGE).
Logarithmically growing A549 cells were transfected in triplicate in
60-mm petri dishes by DEAE-dextran, as previously described (8). Cells were incubated in 2 ml of HEPES-buffered DMEM
(10 mM HEPES, pH 7.4) containing 20 µl of 60 mg/ml DEAE-dextran
(Pharmacia) premixed with 6 µg of RANTES-pGL2 plasmids and 1 µg of
cytomegalovirus-
-galactosidase internal control plasmid. After
3 h, medium was removed, and 0.5 ml of 10% (vol/vol) DMSO in PBS
was added to the cells for 2 min. Cells were washed with PBS and
cultured overnight in 10% fetal bovine serum-DMEM. The next morning,
cells were stimulated with TNF-
(100 ng/ml) and IFN-
(100 IU/ml),
alone or in combination. At different time points, cells were lysed to
measure independently luciferase and
-galactosidase reporter
activity, as previously described (4). Luciferase was
normalized to the internal control
-galactosidase activity. All
experiments were performed in duplicate or triplicate.
EMSA.
Nuclear extracts of untreated and treated A549 cells were prepared
using hypotonic/nonionic detergent lysis, as previously described
(8). Proteins were normalized by protein assay (Protein Reagent, Bio-Rad, Hercules, CA) and used to bind to duplex
oligonucleotides corresponding to the RANTES CRE, ISRE, NF-IL-6, and
NF-
B1 binding sites. Sequences of the oligonucleotides used for EMSA
are shown in Table 1. Nuclear extracts, used for binding to the CRE
site, were prepared from control and treated A549 cells that had been serum-starved before and throughout the period of stimulation for a
total of 24 h.
DNA-binding reactions, using the CRE, ISRE, and NF-IL-6
probes, contained 10-15 µg of nuclear protein, 5% glycerol, 12 mM HEPES, 80 mM NaCl, 5 mM dithiothreitol (DTT), 5 mM
MgCl2, 0.5 mM EDTA, 1 µg of poly(dI-dC), and 40,000 cpm
of 32P-labeled double-stranded oligonucleotide in a total
volume of 20 µl. DNA-binding reactions, using NF-
B1 probe,
contained 10-15 µg of total protein, 5% glycerol, 12 mM HEPES,
80 mM NaCl, 5 mM DTT, 1 µg of poly(dA-dT), and 40,000 cpm of
32P-labeled double-stranded oligonucleotide in a total
volume of 20 µl. The nuclear proteins were incubated with the probe
for 15 min at room temperature and then fractionated by 6%
nondenaturing polyacrylamide gels (PAGE) in TBE buffer (22 mM
Tris · HCl, 22 mM boric acid, and 0.25 mM EDTA, pH 8). After
electrophoretic separation, gels were dried and exposed for
autoradiography using Kodak XAR film at
70°C using intensifying
screens. In competition assays, 2 pmol of unlabeled wild-type (WT) or
mutated competitors were added at the time of probe addition. In the
gel mobility supershift, commercial antibodies (Santa Cruz
Biotechnology, Santa Cruz, CA) against specific transcription factors
were added to the binding reactions and incubated on ice for 1 h
before fractionation on 6% PAGE. In the case of CRE supershift, we
used antibodies broadly reactive with different members of the
activating transcription factor (ATF)/CRE binding (CREB) or activator
protein-1 (AP-1) families. Anti-CREB-1 antibody also recognizes ATF-1
and CRE modulator (CREM)-1 (sc-186); anti-c-Fos recognizes c-Fos,
Fos-B, Fra-1, and Fra-2 (sc-413); anti-c-Jun recognizes c-Jun, Jun-B,
and Jun-D (sc-44). Preimmune serum was used as a control for any
nonspecific effects of the immune antisera.
Microaffinity isolation assay.
Microaffinity purification of proteins binding to the RANTES ISRE was
performed using a two-step biotinylated DNA-streptavidin capture assay
(8). In this assay, duplex oligonucleotides are chemically
synthesized using 5'-biotin on a flexible linker (Sigma Genosys). Four
hundred micrograms of 6-h-treated A549 cell nuclear extracts were
incubated at 4°C for 30 min with 50 pmol of biotin-ISRE, in the
absence or presence of 10-fold molar excess of nonbiotinylated WT or
mutated ISRE. The binding buffer contained 8 µg of poly(dI/dC) (as
nonspecific competitor) and 5% (vol/vol) glycerol, 12 mM HEPES, 80 mM
NaCl, 5 mM DTT, 5 mM MgCl2, and 0.5 mM EDTA. A 50% slurry of prewashed streptavidin-agarose beads (100 µl) was then added to
the sample, which was incubated at 4°C for an additional 20 min with
gentle rocking. Pellets were washed twice with 500 µl of binding
buffer, and the washed pellets were resuspended in 100 µl of 1×
SDS-PAGE buffer, boiled, and fractionated on a 10% SDS-polyacrylamide
gel. After electrophoresis separation, proteins were transferred to
polyvinylidene difluoride membranes for Western blot analysis.
Western immunoblot.
Nuclear proteins were prepared as previously described, fractionated by
SDS-PAGE, and transferred to polyvinylidene difluoride membranes
(8). Membranes were blocked with 5% milk in Tris-buffered saline-Tween and incubated with rabbit polyclonal antibodies to IRF-1
and IRF-3 (Santa Cruz Biotechnology). For secondary detection, we used
a horseradish-coupled donkey anti-rabbit antibody in the enhanced
chemiluminescence assay (Amersham Life Science, Arlington Heights, IL).
Statistical analysis.
Data from experiments involving multiple samples subject to each
treatment were analyzed by the Student-Newman-Keuls t-test for multiple pairwise comparisons. Results were considered
significantly different at P < 0.05.
 |
RESULTS |
TNF-
and IFN-
stimulation induces RANTES gene expression in
A549 cells.
To determine whether cytokine stimulation of A549 cells induces RANTES
protein release, cells were exposed to TNF-
(100 ng/ml) and IFN-
(100 IU/ml), alone or in combination. Cell supernatants were harvested
at different times after stimulation, and RANTES protein secretion was
measured by ELISA. TNF induced a time-dependent release of RANTES from
A549 cells (Fig. 1). IFN-
treatment
alone (shown only for the 24-h time point) did not upregulate RANTES secretion; however, it greatly potentiated the effect of TNF-
. To
determine whether the increased protein release paralleled an increase
in the steady-state level of RANTES mRNA, A549 cells were exposed to
TNF-
(100 ng/ml) and IFN-
(100 IU/ml), alone or in combination,
and total RNA was extracted from control and treated cells at different
time points for Northern blot analysis. A significant increase in
RANTES mRNA expression was detected 3 h after TNF exposure (Fig.
2), with no further increase in mRNA levels at later time points. IFN-
treatment alone did not upregulate RANTES mRNA (data not shown), but it greatly potentiated the effect of
TNF-
. These data indicate that TNF-
and IFN-
synergistically activate RANTES gene expression, which is coupled to protein secretion, in A549 cells.

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Fig. 1.
Effect of tumor necrosis factor (TNF) treatment on
regulated on activation, normal T cell expressed, and presumably
secreted (RANTES) secretion. A549 cells were treated with TNF- (100 ng/ml), alone or in combination with interferon- (IFN- , 100 IU/ml), and culture supernatants were assayed for RANTES production by
ELISA. Values are means ± SD of 3 independent experiments
performed in triplicate.
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Fig. 2.
Northern blot of RANTES mRNA in TNF-treated A549 cells.
Cells were treated with TNF- (100 ng/ml), alone or in combination
with IFN- (100 IU/ml). Total RNA was extracted from control and
infected cells, and 20 µg of RNA were fractionated on a 1.2%
agarose-formaldehyde gel, transferred to a nylon membrane, and
hybridized to a radiolabeled RANTES cDNA probe. Membrane was stripped
and reprobed with -actin.
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TNF-
stimulation induces RANTES promoter activation, whereas
IFN-
stabilizes mRNA transcripts.
To determine whether TNF exposure of alveolar epithelial cells was
inducing RANTES gene expression by activating its transcription, we
transiently transfected A549 cells with a construct containing the
first 974 nt of the human RANTES promoter linked to the luciferase reporter gene (pGL2-974). Previous studies have shown that this fragment of the promoter is sufficient to drive regulated luciferase expression in a variety of cell types (25, 26, 28).
Compared with untreated cells, TNF-
stimulation of transfected A549
cells induced a time-dependent increase of luciferase activity (Fig. 3) that started 3 h after exposure,
peaked at ~6 h, and gradually decreased by 24 h. IFN-
treatment did not induce RANTES promoter activation alone or in
combination with TNF, suggesting that the synergistic effect in RANTES
mRNA induction by the combined stimulation of TNF-
and IFN-
could
be due to an effect of IFN-
on RANTES mRNA stabilization. Therefore,
we investigated the half-life of RANTES mRNA in the presence of IFN-
stimulation. A549 cells were treated with TNF-
, alone or in
combination with IFN-
, for 3 h, and then DRB, an inhibitor of
transcription, was added to the medium. RANTES mRNA levels were
analyzed in cells harvested immediately before addition of DRB (which
represents time 0) and at multiple times thereafter.
Inhibition of transcription caused a progressive decrease in RANTES
mRNA levels in A549 cells treated with TNF-
(Fig.
4), with 90% reduction by 24 h
after termination of transcription. In contrast, in cells treated with
both TNF-
and IFN-
, the decrease in RANTES mRNA levels was much
less pronounced, with a reduction of only ~33% after 24 h
of DRB addition, indicating that IFN-
affects RANTES
mRNA stability. These results indicate that the synergistic
effect of these two cytokines on the upregulation of RANTES expression
in alveolar epithelial cells occurs via the combination of increased
RANTES gene transcription by TNF-
and stabilization of
transcripts by IFN-
.

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Fig. 3.
RANTES promoter activation after treatment with TNF and
IFN. A549 cells were transiently transfected with pGL2-974 plasmid and
treated with TNF- (100 ng/ml) and IFN- (100 IU/ml), alone or in
combination. Cells were harvested to measure luciferase activity.
Uninfected plates served as controls. For each plate, luciferase was
normalized to -galactosidase reporter activity. Values are
means ± SD.
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Fig. 4.
RANTES mRNA half-life after cytokine stimulation. A549
cells were stimulated with TNF- (100 ng/ml), alone or in combination
with IFN- (100 IU/ml), for 3 h, and 100 µM
5,6-dichloro-1- -D-ribofuranosylbenzimidizole (DRB) was
added to treated cells. Total RNA was isolated, and Northern blot
analysis was performed using a radiolabeled RANTES cDNA. RANTES mRNA
was quantitated by exposure of membrane to a PhosphorImager screen and
analysis of images by ImageQuant software. Results are representative
of 1 of 2 separate assays.
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We then focused on characterizing the mechanisms of RANTES promoter
activation after TNF-
stimulation.
Effects of 5'-deletions and site mutations of the RANTES
promoter sequence on TNF-inducible activity.
To define the regions of the RANTES promoter involved in regulating
gene expression after TNF stimulation, A549 cells were transiently
transfected with plasmids containing serial 5'-to-3'-deletions of the
RANTES promoter linked to the luciferase reporter gene. Cells were
treated with 100 ng/ml of TNF-
and harvested 6 h thereafter (conditions corresponding to peak reporter gene induction) to measure
luciferase activity. As shown in Fig. 4, deletions from nucleotides
974 to
220 did not affect basal or TNF-inducible luciferase
activity. Further deletion to nucleotide
150 slightly reduced the
basal activity of the promoter and the TNF-induced luciferase activity
by ~30-40%, indicating that the sequence between nucleotides
220 and
150 is involved in RANTES promoter activation. Deletion to
nucleotide
120 almost completely abolished TNF-induced luciferase
activity, indicating again that the region spanning nucleotides
150
to
120 is important for promoter activation. Recent studies of the
RANTES promoter have identified a CRE site at nucleotides
220 to
190 and an ISRE site in the region at nucleotides
150 to
120 of
the promoter (7, 13, 25). To establish the role of the CRE
and ISRE sites of the RANTES promoter in conferring responsiveness to
TNF stimulation, we tested the effect of point mutations of these sites
in the context of the minimal RANTES promoter fragment (
220 nt) that
retains full TNF inducibility. As shown in Fig.
5, mutation of the CRE site affected basal activity and TNF inducibility of the promoter similar to deletion
of the promoter region spanning nucleotides
220 to
150. Mutation of
the ISRE did not affect the basal activity, but it significantly
reduced TNF-induced promoter activation. Previous studies have
indicated that the NF-IL-6 and two NF-
B binding sites located
between nucleotides
110 and
30 of the promoter can play an
important role in RANTES gene transcription (7, 25, 30).
To determine their role in TNF-induced RANTES transcription, we
introduced site-directed mutations in each of the binding sites, and we
tested the mutant plasmids for TNF inducibility. The proximal NF-
B
site is defined as NF-
B2 and the distal NF-
B site as NF-
B1. As
shown in Fig. 5, mutation of NF-
B1 almost completely abolished TNF-induced promoter activation, whereas mutations of the NF-IL-6 and NF-
B2 sites significantly decreased luciferase activity, although to a lesser extent, indicating that NF-
B1 is the major regulatory site of the RANTES promoter induction after TNF stimulation.

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Fig. 5.
Effects of 5'-deletions and site mutations of the RANTES
promoter sequence on TNF-inducible activity. A: A549 cells
were transiently transfected with 5'-deletions of the human RANTES
promoter and treated with TNF- (100 ng/ml) for 6 h. Uninfected
plates served as controls. For each plate, luciferase was normalized to
-galactosidase reporter activity. Values are means ± SD.
*P < 0.01 vs. pGL2-974. B: A549 cells were
transiently transfected with site-mutated plasmids of the pGL2-220
RANTES promoter and treated with TNF- (100 ng/ml) for 6 h.
Uninfected plates served as controls. For each plate, luciferase was
normalized to -galactosidase reporter activity. WT, wild type; ,
mutation of the indicated promoter site; NF, nuclear factor; IL,
interleukin; CRE, cAMP responsive element; ISRE, interferon-stimulated
responsive element. Values are means ± SD. *P < 0.01 vs. pGL2-220 WT.
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Role of NF-
B and NF-IL-6 in RANTES promoter inducibility after
TNF stimulation.
Because the site mutation analysis of the RANTES promoter had shown
that NF-
B plays a major role in TNF-induced promoter activation, we
performed gel shift assays to determine whether TNF addition produced
changes in the abundance of DNA-binding proteins recognizing this
region of the RANTES promoter.
Two DNA-protein complexes, C1 and C2, were formed from nuclear extracts
of control cells on the NF-
B1 probe (Fig.
6A). TNF exposure markedly
increased the binding of C1 and C2 as early as 1 h after
stimulation, with a decrease in binding intensity at 12 h after
stimulation. The sequence specificity of the different complexes was
examined by competition with unlabeled oligonucleotides in EMSA (Fig.
6B). C1 and C2 were competed by the WT, but not by the
mutated, oligonucleotide, indicating binding specificity. To determine
the composition of the inducible complexes, we performed supershift
assays, adding specific antibodies to various NF-
B subunits in EMSA.
We did not include the antibody anti-RelB, because we and others
previously showed that it is not present in epithelial cells (12,
39). The addition of anti-p50 antibody induced the appearance of
a supershifted band, with a concomitant reduction of C2 intensity (Fig.
6C). Addition of anti-p65 antibody mainly supershifted C1,
indicating that C1 is a p65 homodimer, whereas C2 could be a homo- or
heterodimer of p50.

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Fig. 6.
Electrophoretic mobility shift assay (EMSA) of RANTES
NF- B1 binding complexes in response to TNF treatment. A:
autoradiogram of time course. Nuclear extracts were prepared from
control and TNF-treated cells at 1, 3, 6, and 12 h and used for
EMSA. B: competition analysis. Nuclear extracts from A549
control cells or A549 cells treated with TNF for 6 h were used to
bind to the NF- B1 probe in the absence ( ) or presence of 2 pmol
of unlabeled WT or mutated (MUT) competitor in the binding reaction.
C: supershift assay. Nuclear extracts of A549 cells treated
with TNF for 6 h were used in the EMSA in the presence of control
serum, anti-p50, anti-p52, anti-c-Rel, and anti-p65 antibodies. Arrows,
supershifted bands.
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Similar experiments were performed using the RANTES NF-IL-6 binding
site. When nuclear extracts from A549 cells were used to bind to the
NF-IL-6 probe, three complexes (C1, C2, and C3) were formed in control
cells (Fig. 7A). TNF exposure
markedly increased the binding of C1 and C3 as early as 1 h after
stimulation, which remained similar up to 12 h after stimulation.
In a competition assay, C1 and C3 were competed by the WT, but not
by the mutated, oligonucleotide, indicating binding specificity (Fig.
7B). In a supershift assay, using antibodies
anti-CCAAT/enhancer binding protein (C/EBP)-
, anti-C/EBP
/NF-IL-6,
and anti-C/EBP
, the main C/EBP family members involved in promoter
transactivation (31), the addition of anti-C/EBP
resulted in the disappearance of C1 and C3 nucleoprotein complexes and
the appearance of a supershifted band (arrow), showing that C/EBP
is
the major component of the TNF-inducible C1 and C3 (Fig.
7C). Together, the EMSA indicates that TNF stimulation
induced significant changes in the composition of proteins binding to
the proximal RANTES NF-IL-6 and NF-
B sites.

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Fig. 7.
EMSA of RANTES NF-IL-6 binding complexes in response to
TNF treatment. A: autoradiogram of time course. Nuclear
extracts were prepared from control and TNF-treated cells at 1, 3, 6, and 12 h and used for EMSA. B: competition analysis.
Nuclear extracts from untreated (control) A549 cells and A549 cells
treated with TNF for 6 h were used to bind to the NF-IL-6 probe in
the absence ( ) or presence of 2 pmol of unlabeled WT or mutated
competitor. C: supershift assay. Nuclear extracts of A549
cells treated with TNF for 6 h were used in the EMSA in the
presence of control serum, anti-CCAAT/enhancer binding protein
(C/EBP)- , anti-C/EBP , and anti-C/EBP . Arrow, supershifted
band.
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Role of CRE and ISRE binding proteins in RANTES promoter
inducibility after TNF stimulation.
To determine whether TNF stimulation produced changes in the abundance
of DNA-binding proteins recognizing the RANTES CRE site, EMSA was
performed on nuclear extracts prepared from control and TNF-treated
A549 cells. The CRE sites can bind homo- and heterodimeric complexes
formed by members of the CREB, ATF, Jun, and Fos transcription factor
families. To exclude uncontrolled effects due to the presence of serum
on c-Fos and Jun expression (20), serum-starved cells were
stimulated with TNF-
(100 ng/ml) and harvested at different times to
prepare nuclear extracts. Two binding complexes, C1 and C2, were
detected in control A549 cells (Fig.
8A); TNF treatment slightly
increased C1 binding, which could be detected 1 h after stimulation and persisted for the duration of the experiment. The
inducible C1 complex was sequence specific, as demonstrated by its
competition by an unlabeled WT, but not a mutant, oligonucleotide (Fig.
8B). To determine the composition of the TNF-inducible
complex, we performed supershift assays using a panel of antibodies
broadly reacting with the different members of CREB, ATF, Fos, and Jun families of transcription factors (see METHODS). Addition
of anti-CREB-1 antibody caused a significant reduction of C1 complex,
whereas addition of anti-ATF-2 antibody induced the appearance of a
faint supershifted band (Fig. 8C, arrow), suggesting that
these members of the CREB/ATF family are the major components of the
CREB complexes.

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Fig. 8.
EMSA of RANTES CRE binding complexes in response to TNF
stimulation. A: autoradiogram of time course. Nuclear
extracts were prepared from control and TNF-treated cells at 1, 3, 6, and 12 h and used for EMSA. B: competition analysis.
Nuclear extracts from untreated (control) A549 cells and A549 cells
treated with TNF for 6 h were used to bind to the CRE probe in the
absence ( ) and presence of 2 pmol of WT and mutated unlabeled
competitor in the binding reaction. C: supershift assay.
Nuclear extracts of A549 cells treated with TNF for 6 h were used
in the EMSA in the presence of control serum, anti-Jun, anti-Fos,
anti-CREB-1, anti-CREB-2, and anti-activating transcription factor
(ATF)-2 antibodies. Arrow, supershifted band.
|
|
Finally, we investigated changes in the abundance of DNA-binding
proteins recognizing the RANTES ISRE site after TNF treatment. Three
nucleoprotein complexes, C1, C2, and C3, were formed in control cells
using the ISRE probe (Fig.
9A). TNF stimulation increased
the binding of C2 and C3 as early as 30 min after exposure, with a peak
in binding intensity 6 h after TNF stimulation (binding intensity
decreased at later time points; data not shown). The sequence
specificity of the ISRE complexes was examined by competition with
unlabeled oligonucleotides in EMSA (Fig. 9B). C2 and C3 were competed by the WT, but not by the mutated, oligonucleotide, indicating binding specificity. RANTES ISRE binds transcription factors belonging to the ISRE family. To determine the composition of the TNF-inducible complex, preimmune serum or antibodies recognizing IRF-1, IRF-2, IRF-3,
and IRF-7 were added to the binding reaction. We did not include
anti-interferon consensus sequence binding protein, because this
protein is expressed only in hematopoietic cell types
(37). The anti-IRF-1 antibody affected C2 and C3 binding
(Fig. 9C), inducing the disappearance of the complexes and
the appearance of a supershifted band (arrow), indicating that IRF-1 is
a major component of the TNF-inducible complexes.

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Fig. 9.
EMSA of RANTES ISRE binding complexes in response to TNF
stimulation. A: autoradiogram of time course. Nuclear
extracts were prepared from control and TNF-treated cells at 0.5, 1, 3, and 6 h and used for EMSA. B: competition analysis.
Nuclear extracts from untreated (control) A549 cells and A549 cells
treated with TNF for 6 h were used to bind to the ISRE probe in
the absence ( ) and presence of 2 pmol of unlabeled WT and mutated
competitor. C: supershift assay. Nuclear extracts of A549
cells were treated with TNF for 6 h in the presence of control
serum, anti-interferon regulatory factor (IRF)-1, anti-IRF-2,
anti-IRF-3, and anti-IRF-7 antibodies. Arrow, supershifted band.
|
|
We and others previously showed that IRF-3 and IRF-7 play an
important role in virus-induced RANTES gene transcription
(7, 13). Because the absence of a supershift cannot be
used to exclude the presence of IRF-3 or IRF-7 binding to the ISRE, we
first investigated whether TNF stimulation was able to induce IRF-3
activation. Nuclear extracts prepared from A549 control cells and A549
cells treated with TNF for various lengths of time were used for
Western blot analysis. TNF stimulation induced a time-dependent nuclear
accumulation of IRF-1 and IRF-3 starting at ~3 h after exposure
(Fig. 10A). We then used a
two-step microaffinity isolation-Western blot to determine whether
IRF-3 was binding to the RANTES ISRE after TNF stimulation. In this
assay, biotinylated ISRE was used to bind nuclear extracts of control
A549 cells and A549 cells treated with TNF for 6 h. ISRE binding
proteins were captured by the addition of streptavidin-agarose beads
and washed, and the presence of bound IRF-1 and IRF-3 was detected by
Western blot. As a control, we used nuclear extracts prepared from A549
cells infected with RSV, with a multiplicity of infection of 1, for
12 h, which we previously showed is able to induce IRF-1 and IRF-3
binding to the RANTES ISRE (7). There was little binding
of IRF-1 and no binding of IRF-3 in control nuclear extracts (Fig.
10B). IRF-1 binding was greatly increased after TNF
stimulation and RSV infection. However, although IRF-3 from A549 cells
infected with RSV was able to bind to the RANTES ISRE, IRF-3 from
TNF-stimulated cells did not. IRF-1 and IRF-3 detection was abolished
when 10-fold excess of ISRE oligonucleotide was included as competitor
in the initial binding reaction, indicating sequence specificity. These data indicate that although TNF stimulation induces IRF-1 and IRF-3
nuclear translocation, only IRF-1 binds to the RANTES ISRE site.
Finally, we investigated whether TNF stimulation was able to activate a
promoter driven by the RANTES ISRE. A549 cells were transfected with a
reporter gene containing multimers of the RANTES ISRE and infected with
RSV or stimulated with TNF-
for various lengths of time. Activity of
the ISRE multimer was highly inducible after RSV infection but was not
significantly affected by TNF stimulation (Fig.
11), indicating that RANTES gene
enhancers are controlled in a stimulus-specific fashion.

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Fig. 10.
Western blot and microaffinity isolation of IRF-1 and
IRF-3 protein in A549 cells treated with TNF. A: A549 cells
were stimulated with TNF- (100 ng/ml) and harvested at 0, 3, 6, and
12 h for preparation of nuclear extracts. Equal amounts of protein
from control and treated cells were assayed for IRF-1 and IRF-3 by
Western blot. B: nuclear extracts were prepared from
untreated (control) A549 cells and A549 cells stimulated with TNF (100 ng/ml) for 6 h or infected with respiratory syncytial virus (RSV,
multiplicity of infection = 1) for 12 h. IRF proteins were
affinity purified using biotinylated ISRE in the absence ( ) or
presence (+) of nonbiotinylated WT competitor. After capture with
streptavidin-agarose beads, complexes were eluted and assayed for IRF-1
and IRF-3 by Western blot.
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|

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Fig. 11.
RANTES ISRE-driven promoter activation after viral
infection and TNF stimulation. A549 cells were transiently transfected
with multimers of the RANTES ISRE linked to a luciferase reporter gene.
Cells were infected with RSV at multiplicity of infection of 1 or
stimulated with TNF- (100 ng/ml) for 3, 6, and 15 h. Cells were
harvested to measure luciferase activity. Uninfected plates served as
controls. For each plate, luciferase was normalized to
-galactosidase reporter activity. Values are means ± SD.
|
|
 |
DISCUSSION |
The respiratory epithelium represents the principal cellular
barrier between the environment and the internal milieu of the airways
and is responsible for particulate clearance and surfactant secretion.
After exposure to infectious agents, allergens, chemical pollutants,
and particulate matter, airway epithelial cells are able to secrete a
variety of proinflammatory molecules, including chemokines, which are
responsible for the selective recruitment of circulating leukocytes
into the lung (16, 18). RANTES is a member of the CC
branch of the chemokine family, strongly chemotactic for T lymphocytes,
monocytes, basophils, and eosinophils (1, 34), and has
been implicated in a variety of diseases characterized by inflammation.
Intercellular communication by cytokines during an inflammatory
reaction is important to its orchestration and subsequent resolution.
IFN-
and TNF-
are pleiotropic cytokines that often play a
critical role in this process. Human RANTES gene expression appears to
be differentially regulated depending on the cell type and the stimulus
applied (17, 25, 26, 28, 30). Because identification of
the regulatory mechanisms involved in RANTES gene transcription is
important for a rational design of therapeutic agents that can block
its expression in the lung, in this study we have examined the
requirements for activation of RANTES gene expression after cytokine stimulation.
Our results show that IFN-
and TNF-
synergistically induce RANTES
protein secretion and gene expression in alveolar epithelial cells
(Figs. 1 and 2). However, the synergism does not occur at the level of
RANTES gene transcription, because TNF-
is able to induce the RANTES
promoter-driven expression of the luciferase reporter gene but not
IFN-
(Fig. 3). Treatment of alveolar epithelial cells with IFN-
results in a prolonged half-life of RANTES transcripts (Fig. 4),
indicating that the synergism occurs through RANTES mRNA stabilization,
which has been shown to play an important role in virus-induced RANTES
gene expression (19). Our results obtained in alveolar
epithelial cells are different from results recently shown in
fibroblasts, in which IFN-
and TNF-
stimulation synergistically
activated the murine RANTES promoter (21), but are similar
to results reported in astrocytes and glial cells after IL-1
or
TNF-
and IFN-
stimulation, which synergize in RANTES protein
secretion and gene expression, but not transcription (22,
25). In glial cells, the half-life of RANTES mRNA in the
presence of TNF-
and IFN-
was almost double that in the presence
of TNF-
alone (22), which is very similar to our
findings in alveolar epithelial cells.
Results from 5'-deletion and mutation analysis indicate that several
cis-regulatory elements are required for full TNF-induced promoter activation. The shortest RANTES promoter fragment that is
activated similarly to the full-length promoter after TNF-
stimulation comprises the first 220 bp from the start codon. Deletion of the region spanning nucleotides
220 to
195 reduces promoter inducibility ~50%, and a second deletion from nucleotides
150 to
120 almost completely abolishes it (Fig. 4). A few studies have
investigated the minimal enhancer region of the RANTES promoter required to confer responsiveness to external stimuli such as cytokines
or phorbol esters. In T cell and monocyte-like cell lines, the first
500 nucleotides of RANTES promoter are sufficient to confer
full phorbol 12-myristate 13-acetate and ionomycin inducibility, which
is lost by further deletions to nucleotide
200 (26). Differently, in phytohemagglutinin-stimulated lymphocytes, a
195-bp fragment of RANTES promoter has the same inducibility as
longer fragments, whereas a 120-bp fragment is no longer inducible
(28, 30). In a human astrocytoma cell line, IL-1
stimulation of RANTES promoter requires a region spanning nucleotides
220 to
120 (25), similar to our findings in alveolar
epithelial cells infected with RSV (7), indicating that
the minimal enhancer region required for RANTES promoter activation
differs among cell types.
The site mutation analysis indicates that the CRE, ISRE, NF-IL-6, and
NF-
B sites play an important role in TNF-induced RANTES promoter
activation (Fig. 5), which is similar to our findings in alveolar
epithelial cells infected with RSV (7). However, there are
some important differences in the relative importance of these
cis-regulatory elements in RANTES induction, as well as in
the type of transcription factors binding to them, after these two
different stimuli. The CRE site accounts for 40-50% of
TNF-induced promoter activation, as shown by the deletion and site
mutation analysis, which is similar to our findings in virus-infected cells (7). However, although TNF-
stimulation induces
binding to the CRE site of transcription factors belonging to the CREB and ATF families (Fig. 8C), RSV infection also induces
activation and binding of c-Jun (7).
Mutation of the NF-
B1 site almost completely abolishes TNF-induced
promoter activity, indicating that the NF-
B site plays a fundamental
role in cytokine-induced RANTES gene transcription. This result is
different from our previous finding in RSV-infected cells, in which
mutation of the NF-
B1 site accounts for a ~60% reduction of the
promoter inducibility, although the composition of the DNA-nuclear
complexes formed on the RANTES NF-
B1 site is identical in cytokine
and viral stimulation, containing p65 and p50 subunits (Fig.
6C).
The situation is reversed for the ISRE site, since its mutation reduces
TNF-stimulated luciferase activity ~50%, whereas it completely
abolishes luciferase inducibility after RSV infection (7), indicating that the ISRE site is absolutely necessary for virus-induced RANTES promoter activation, but not for
cytokine stimulation. Furthermore, the composition of the
nucleoprotein complexes binding to the ISRE site after TNF-
addition
or viral infection is quite different. Supershift assays and
microaffinity isolation experiments (Figs. 9C and
10B) clearly show that TNF-
stimulation induces IRF-1,
but not IRF-3, binding to the RANTES ISRE, even if it is able to induce
IRF-3 translocation to the nucleus (Fig. 10A). This result
is very different from our finding in virus-infected epithelial cells.
RSV infection of A549 cells induces IRF-1, IRF-3, and IRF-7 binding to
the ISRE site (8), similar to the finding of Lin et
al. (23) in Sendai virus-infected cells. IRF-3
binding to the ISRE is necessary for activation of the ISRE
site, as demonstrated by the inability of TNF-
to stimulate a RANTES
ISRE-driven promoter, which is highly inducible after viral infection
of alveolar epithelial cells (Fig. 11), indicating that RANTES gene
enhancers are controlled in a stimulus-specific fashion.
In conclusion, our findings indicate that cooperation among
transcription factors belonging to the C/EBP, NF-
B, IRF, and CREB/ATF families is necessary for full transcriptional activation of
the RANTES promoter after cytokine stimulation, similar to our finding
in virus-infected cells, supporting again the "enhanceosome" model for RANTES gene transcription. However, the type and role of the
different families of transcription factors in RANTES promoter activation in cytokine-stimulated cells are different from those in
virus-infected cells, emphasizing the importance of detailed analysis
of the mechanisms regulating tissue- and stimulus-specific expression
of important genes, such as RANTES, to develop strategies for
modulating the host immune/inflammatory responses in the lung.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by National Institutes of Health
Grants AI-01763, P01 AI-46004, and P30 ES-06676 and a Beginning Grant-in-Aid of the American Heart Association (to A. Casola).
 |
FOOTNOTES |
Address for reprint requests and other correspondence: A. Casola, Dept. of Pediatrics, Div. of Child Health Research Center, 301 University Blvd., Galveston, TX 77555-0366 (E-mail:
ancasola{at}utmb.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.
August 9, 2002;10.1152/ajplung.00162.2002
Received 23 May 2002; accepted in final form 3 August 2002.
 |
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