From the Section of Pulmonary and Critical Care
Medicine, Yale University School of Medicine, Department of Internal
Medicine, New Haven, Connecticut 06520-8057 and § Indiana
University School of Medicine, Departments of Medicine
(Hematology/Oncology), and Biochemistry/Molecular Biology, Walther
Oncology Center, Indianapolis, Indiana 46202
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ABSTRACT |
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Studies were undertaken to characterize the
mechanism by which transforming growth factor-1
(TGF-
1) stimulates epithelial cell interleukin (IL)-11
production. Nuclear run-on studies demonstrated that
TGF-
1 is a potent stimulator of IL-11 gene
transcription. TGF-
1 also stimulated the luciferase
activity in cells transfected with reporter gene constructs containing
nucleotides
728 to +58 of the IL-11 promoter. Studies with
progressive 5' deletion constructs and site-specific mutations
demonstrated that this stimulation was dependent on 2 AP-1 sites
between nucleotides
100 and
82 in the IL-11 promoter. Mobility
shift assays demonstrated that TGF-
1 stimulated AP-1
protein-DNA binding to both AP-1 sites. Supershift analysis
demonstrated that JunD was the major moiety contributing to AP-1-DNA
binding in unstimulated cells and that c-Jun-, Fra-1-, and Fra-2-DNA
binding were increased whereas JunD-DNA binding was decreased in
TGF-
1-stimulated cells. The sequence in the IL-11
promoter that contains the AP-1 sites also conferred TGF-
1 responsiveness, in a position-independent fashion,
on a heterologous minimal promoter. Thus, TGF-
1
stimulates IL-11 gene transcription via a complex
AP-1-dependent pathway that is dependent on 2 AP-1 motifs
between nucleotides
100 and
82 that function as an enhancer in the
IL-11 promoter.
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INTRODUCTION |
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Interleukin-11 (IL-11)1 was originally discovered as a soluble factor in supernatants from transformed stromal cells that stimulated plasmacytoma cell proliferation (1). It has subsequently been shown to be a pleiotropic member of the IL-6-type cytokine family that mediates its biologic activities via binding to a multimeric receptor complex that contains the gp130 molecule (2-5). Among its many effects are the ability to regulate hematopoiesis, stimulate the production of acute phase proteins, induce the tissue inhibitor of metalloproteinase-1, regulate bone metabolism, and alter epithelial proliferation (2, 6-10). Studies from our laboratories and others have also demonstrated that IL-11 can induce tissue fibrosis, regulate tissue myocyte and myofibroblast accumulation, alter airway physiology, and confer protection in the context of mucosal injury of the respiratory and gastrointestinal tracts (11-14).
In keeping with the biologic importance of IL-11, a number of
investigators have studied its sites of production and the regulation of these responses. These studies demonstrated that IL-11 is produced by a variety of stromal cells in response to a variety of stimuli, including cytokines, histamine, eosinophil major basic protein, and
respiratory tropic viruses (7, 15-20). A prominent finding in our
studies of fibroblasts (18), epithelial cells (19), and osteoblasts
(20) and studies by others of chondrocytes and synoviocytes (7) has
been the importance of TGF- moieties in the stimulation of IL-11
production. These studies also demonstrated that TGF-
1
stimulation of IL-11 protein production is associated with
proportionate changes in IL-11 mRNA accumulation and, in our
studies, IL-11 gene transcription (18).
The IL-11 promoter has been cloned and the cis-elements and
trans-acting factors that regulate the levels of basal IL-11 production have been identified by our laboratories (21). Despite the demonstrated importance of gene transcription in the stimulation of IL-11
production, the cis-elements and trans-acting factors that mediate the
transcriptional activation of IL-11 have not been investigated. To
further our understanding of the regulation of IL-11, studies were
undertaken to characterize the transcriptional elements utilized by
TGF-1 in the stimulation of IL-11. These studies
identify two activating protein-1 (AP-1) motifs between
100 and
82
in the IL-11 promoter that are essential for TGF-
-induced IL-11
transcriptional activation. They also demonstrate that this stimulation
is associated with complex alterations in the composition of the AP-1
subunits that bind to these sites and that DNA which contains these
AP-1 elements confers TGF-
1 responsiveness on a
heterologous promoter. Lastly they demonstrate that this mechanism is
stimulus-specific since respiratory syncytial virus (RSV) stimulates
IL-11 transcription via a different mechanism.
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EXPERIMENTAL PROCEDURES |
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Cell Culture and TGF-1 Stimulation
A549 human alveolar epithelial-like cells were obtained from the
American Type Culture Collection (ATCC, Rockville, MD) and grown to
confluence in Dulbecco's modified Eagle's medium (DMEM) supplemented
with 10% fetal bovine serum (22). At confluence, varying
concentrations of recombinant human TGF-1 (1-10 ng/ml) (R & D Systems, Minneapolis) or medium controls were added, and the
cells were incubated for up to 48 h. At the desired points in
time, supernatants were removed and stored at
20 °C, and nuclei were harvested for further usage (see below).
Nuclear Run-on Assay
The relative rates of gene transcription were assessed using
modifications of protocols previously described by our laboratory (22-25). A549 cells were incubated for 16 h under control
conditions, with TGF-1 (10 ng/ml), or after infection
with respiratory syncytial virus (RSV) at a multiplicity of infection
of 3 as described previously (17-19). The cells (3 × 107 per condition) were then washed twice with ice-cold
phosphate-buffered saline, pelleted, and resuspended in lysis buffer
(10 mM Tris-HCl, pH 7.4, 2 mM
MgCl2, 3 mM CaCl2, 3 µM dithiothreitol (DTT), 300 mM sucrose,
0.5% Triton X-100). The nuclei were then harvested by centrifugation
and resuspended in 100 µl of storage buffer (50 mM
Tris-HCl, pH 8.3, 5 mM MgCl2, 0.1 mM EDTA, 40% glycerol) and stored at
80 °C until
further utilized. Nylon membranes were prepared carrying 20 µg each
of isolated cDNA fragments encoding IL-11 (a gift of Dr. Paul
Schendel, Genetics Institute, Cambridge, MA) and pUC18 without a
cDNA insert (a control for nonspecific hybridization) using a
slot-blotting apparatus (MINI-FOLD II, Schleicher & Schuell) and baked
in a vacuum oven (80 °C for 2 h). When ready, nuclei were
thawed on ice and pelleted in a microcentrifuge at 4 °C for 30 s, and in vitro transcription and RNA labeling were carried
out in transcription buffer (20 mM Tris-HCl, pH 8.3, 100 mM KCl, 4.5 mM MgCl2, 2 mM DTT, and 400 µM each of ATP, GTP, and CTP)
in the presence of 200 µCi of [
-32P]UTP (~3000
Ci/mmol, Amersham Corp.) and 20% glycerol at 30 °C for 30 min. The
reaction was followed with a cold chase with 1 µl of 100 mM UTP for 10 min at 30 °C. The reaction was then
terminated by incubating with stop buffer (50 mM Tris-HCl,
pH 8.3, 500 mM NaCl, 5 mM EDTA) with 200 µg/ml RNase-free DNase I and 750 units/ml RNasin (Boehringer
Mannheim) at 30 °C for 15 min. RNA was extracted with
phenol/chloroform, precipitated, and washed with alcohol. Dried RNA
pellets were dissolved in equal volumes of TE buffer (10 mM
Tris-HCl and 1 mM EDTA, pH 7.8), and radioactivity was determined by the mean of duplicate countings of 1-µl aliquots. Hybridization was performed by incubating each membrane with equal numbers of counts of radiolabeled RNA. The membranes were then washed
at high stringency, and binding was evaluated using
autoradiography.
Primer Extension Analysis
A549 cells were incubated for 16 h with
TGF-1 (10 ng/ml). The supernatants were then removed,
and poly(A)+ RNA was isolated using oligo(dT) affinity
based methodology as described (26, 27). Primer extension was then
performed using a radiolabeled 20-base complementary synthetic
oligonucleotide corresponding to oligonucleotides
11 to +9 with
respect to the translation start site. The 5' end of the resulting IL-1
mRNA was defined using the Moloney murine leukemia virus primer
extension system (Promega, Madison, WI) as described by the
manufacturer. In this system the 5' end-labeled oligonucleotide
hybridized with the IL-11 mRNA and was utilized as a primer by the
Moloney murine leukemia virus reverse transcriptase which, in the
presence of deoxynucleotides, synthesized cDNA until the 5' end of
the mRNA was reached. The extended product was then resolved on an
8% urea/polyacrylamide sequencing gel along with a known DNA sequence
ladder.
Plasmid Construction
A 786-bp PvuII fragment of the human IL-11 promoter
was previously isolated and cloned in our laboratories (21). This
promoter fragment contained the sequences between 728 and +58
relative to the transcription start site defined above. It was cloned
into the SmaI site of the luciferase reporter gene vector
pXP2-luc (ATCC) to generate the construct pXP2-IL-11-728.
Preparation of 5' Deletion Constructs
Two techniques were used to generate a series of 5' deletions of
the pXP2-IL-11-728 parent construct. When appropriate restriction sites
were present, they were utilized to generate deletion mutants. This
approach was utilized with the AvaII site at 324 and the HinfI site at
96. In both cases, the
728 to +58 fragment
of the IL-11 promoter was subjected to enzyme digestion, and the desired fragment was recloned into the vector pXP2-luc using standard approaches. When appropriate restriction sites were not available Bal-31 exonuclease digestion was employed to introduce deletions. This
technique takes advantage of the fact that Bal-31 degrades both the 5'
and 3' ends of double-stranded DNA without inserting internal cleavages
(28). Briefly, BamHI-linearized parent construct pXP2-IL-11-324 was incubated with Bal-31 exonuclease for varying periods. BamHI linkers (New England Biolabs,
catalogue number 1071, Beverly, MA) were then added, and the DNA was
subjected to BamHI/XhoI double digestion. The DNA
fragments with the various 5' deletions were then separated by
electrophoresis, electroeluted, and ligated into
BamHI/XhoI-linearized pXP2-luc vector. Clones from the subsequent transformation were screened for insert size, and
DNA sequencing was used to verify junction sequences for all clones
that were chosen for further utilization.
Through the combined efforts of both approaches, a series of
constructs were prepared whose 5' ends extended from 728 to
81. In
all cases, the 3' end was +58 relative to the transcription initiation
site.
Site-directed Mutagenesis
Mutation of the AP-1 sites in the parent IL-11 promoter was
performed using the Muta-Gene M13 In Vitro Mutagenesis Kit
(Bio-Rad, catalog number 170-3580) based on Kunkel's method (29, 30). The 382-bp BamHI/XhoI fragment of IL-11 promoter
was excised from pXP2-IL-11-324 and subcloned into M13 phage. The
recombinant phage DNA was then transformed into bacterial strain
Escherichia coli CJ236 (dut, ung
, thi
, and relA
) to
generate uracil-containing single-stranded DNA. Such single-stranded
DNA was allowed to anneal to mutagenic primer, and second strand DNA
was synthesized with T7 DNA polymerase and T4 DNA ligase. When
transformed into bacterial strain MV1190(dut+, ung+), uracil-containing
single-stranded DNA template was degraded, and only newly synthesized
mutation-bearing second strand DNA would propagate. The wild type and
mutated AP-1 sequences are as follows: wild type 5' (distal) AP-1,
5'-TGAGTCA-3'; mutated 5' (distal) AP-1, 5'-TGAcgaA-3'; wild type 3'
(proximal) AP-1, 5'-TGTGTCA-3'; mutated 3' (proximal) AP-1,
5'-TGTcgaA-3'. All of the AP-1 mutation constructs underwent DNA
sequencing to verify the site and extent of the induced
alterations.
Preparation of IL-11/tk/luc Constructs
A 156-bp BamHI/XhoI fragment from the
herpes simplex virus thymidine kinase (tk) minimal
promoter/chloramphenicol acetyltransferase reporter gene construct
ptk-CAT (31) was obtained from Dr. Anuradha Ray (Yale University, New
Haven) and subcloned into the BamHI and XhoI
sites of the pXP2-luc reporter gene construct to generate ptk-XP2. Two
oligonucleotides (5'-GAT CCG AGG GTG AGT CAG GAT GTG TCA GGC CGA AGC
TT-3' and 5'-GAT CAA GCT TCG GCC TGA CAC CTG ACT CAC CCT CG-3') were
then synthesized and annealed to form a 38-bp DNA duplex with sticky
ends compatible with the BamHI site (5' of herpes simplex
virus tk promoter) in ptk-XP2. This insert contains a 27-bp DNA
sequence of the IL-11 promoter (103 to
77 relative to transcription
start site) that contains both the 5' and 3' AP-1 sites. This
double-stranded DNA sequence was then cloned into ptk-XP2 in the
correct (sense) (pIL11(+)tk-XP2) and reverse (antisense)
(pIL11(
)tk-XP2) directions. DNA sequencing was performed to verify
the sequence and orientation of DNA insertion.
Cell Transfection and Reporter Gene Assay
A549 cells were seeded at 40-50% confluence and incubated
overnight in DMEM with high glucose and 10% fetal bovine serum. Transfections were performed using the DEAE-dextran method as described
previously by our laboratory (22). The cells were then incubated for
24 h in serum-free DMEM alone or in DMEM supplemented with
TGF-1 (10 ng/ml). In experiments where RSV was utilized the A549 cells were incubated for 90 min with RSV (multiplicity of
infection = 3) or appropriate medium control, washed, and then incubated for 24 h in serum-free DMEM. At the end of these
incubations, cell lysates were prepared, and luciferase activity was
assessed on a Lumat luminometer using the Luciferase Assay System from Promega (Madison, WI). In all transfections the construct pCMV-
-gal (CLONTECH, Palo Alto, CA) was also included to
control for transfection efficiency. The
-galactosidase activity in
unstimulated and stimulated cell lysates was characterized using the
CPRG method as described previously by this laboratory (22). The
-galactosidase levels were then used to standardize the measurements
of luciferase activity.
Electrophoretic Mobility Shift Assay (EMSA)
Preparation of Nuclear Extracts--
Nuclear extracts were
prepared using modifications of the techniques of Schreiber et
al. (32). Unstimulated, TGF-1-stimulated, and
RSV-infected A549 cells were prepared as noted above. At the desired
points in time, the cells (107 per condition) were
mechanically detached, suspended in Tris-buffered saline freshly
supplemented with protease inhibitors (1 µg/ml leupeptin, 5 µg/ml
aprotinin, and 1 mM phenylmethylsulfonyl fluoride), pelleted at 4 °C, and resuspended, and swollen in solution A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM
EDTA, 0.1 mM EGTA, 1 mM DTT with freshly added
protease inhibitors as above) for 15 min on ice. Membrane lysis was
accomplished by adding 25 µl of Nonidet P-40 followed by vigorous
agitation. The nuclei were collected by centrifugation, resuspended in
50 µl of solution B (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT and freshly added protease inhibitors as above), and
agitated vigorously at 4 °C for 15 min. The membrane debris was
discarded, and the nuclear extracts were snap-frozen in small aliquots
and stored at
80 °C. The protein concentrations of the nuclear
extracts were determined using the DC Protein Assay System
(Bio-Rad).
Oligonucleotide Probes-- Double-stranded oligonucleotide probes were used in these experiments. For the sake of simplicity, only the top strand DNA sequences are illustrated here. Four oligonucleotide probes were synthesized using the oligonucleotide synthesis facility at Yale University. They include the following: (i) wild type 5' AP-1 sequence in the IL-11 promoter (5' AP-1) (5'-GGGAGGGTGAGTCAGGATGTG-3'); (ii) mutated 5' AP-1 (5'-GGGAGGGTGAcgaAGGATGTG-3'); (iii) wild type 3' AP-1 sequence in the IL-11 promoter (3' AP-1) (5'-AGTCAGGATGTGTCAGGCCGGCCC-3'); and (iv) mutated 3' AP-1 (5'-AGTCAGGATGTcgaAGGCCGGCCC-3').
Four other oligonucleotides were obtained from commercial sources (Stratagene, La Jolla, CA). They included the following: (i) a classic AP-1 oligonucleotide (5'-CTAGTGATGAGTCAGCCGGATC-3'); (ii) an AP-2 oligonucleotide (5'-GATCGAACTGACCGCCCGCGGCCCGT-3'); (iii) an AP-3 oligonucleotide (5'-CTAGTGGGACTTTCCACAGATC-3'); and (iv) an SP-1 oligonucleotide (5'-GATCGATCGGGGCGGGGCGATC-3').Electrophoresis--
EMSAs were performed using the techniques
of Schreiber et al. (32). Radiolabeled double-stranded
oligonucleotide probes were prepared by annealing complementary
oligonucleotides and end-labeling using [-32P]ATP and
T4 polynucleotide kinase (New England Biolabs). The labeled probes were
purified by push-column chromatography, diluted with TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) to the desired concentration, and incubated with equal aliquots of nuclear extract (2-5 µg) and 2 µg of poly[dI-dC]·poly[dI-dC] in a total
volume of 20 µl at room temperature for 1 h. Resolution was
accomplished by electrophoresing 10 µl of the reaction solution on
vertical 6% native polyacrylamide gels containing 2% glycerol using
25% TBE buffer (22.3 mM Tris-HCl, 22.3 mM
boric acid, 0.25 mM EDTA, pH 8.0). Binding was assessed via
autoradiography.
Supershift EMSA--
Supershift assays were used to determine
which members of the AP-1 family were involved in
TGF1-stimulation of IL-11 gene transcription. In these
studies EMSA were performed as described above except that isotype
matched rabbit polyclonal antibodies against AP-1 proteins or control
preimmune antiserum were included during the 1-h radiolabeled
probe-extract incubation period. All of the antibodies that were used
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). They
included antibodies that react with all Jun family members (Pan-Jun)
(catalogue number SC-44X), JunB (catalogue number 46X), JunD (catalogue
number SC-74X), c-Jun (catalogue number SC822X); all Fos family members
(pan-Fos) (catalogue number SC-253X), c-Fos (catalogue number SC-52X),
FosB (catalogue number SC-48X), Fra-1 (catalogue number SC-605X), and Fra-2 (catalogue number SC604X).
Respiratory Syncytial Virus (RSV) Preparation and Infection
RSV (A-2 strain) was obtained from the ATCC. Stock virus was prepared in permissive cell lines and titered, and A549 cells were infected with the virus as described previously by this laboratory (17, 19).
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RESULTS |
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TGF-1 and RSV Stimulate IL-11 Gene
Transcription--
Previous studies from our laboratory demonstrated
that TGF-
1 is a potent stimulator of IL-11 gene
transcription in lung fibroblasts (18) and that TGF-
1
and RSV stimulate A549 alveolar epithelial cell IL-11 protein
production and mRNA accumulation (17, 19). To determine if both
stimuli augment IL-11 gene transcription in A549 cells, nuclear run-on
assays were performed, and the levels of IL-11 gene transcription were
evaluated at base line, after TGF-
1 stimulation and
after RSV infection. At base line, the levels of IL-11 gene
transcription in A549 cells were near the limits of detection with our
assay (Fig. 1). In contrast, both TGF-
1 and RSV caused significant increases in IL-11 gene
transcription (Fig. 1).
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Characterization of the Transcription Start Site in the IL-11
Promoter--
Prior to initiating studies designed to define the
stimulation-responsive cis-elements in the IL-11 promoter, primer
extension analysis was used to characterize the transcription
initiation site in TGF-1-stimulated A549 cells. A single
transcription initiation site was detected. This start site was 154 bp
upstream of the ATG (data not shown) and is within 1-2 bases of the
start site previously described in unstimulated PU-34 primate bone
marrow fibroblasts by our laboratories (21).
TGF-1 Stimulates IL-11 Promoter Activity--
To
begin to characterize the mechanism by which TGF-
1
stimulates IL-11 gene transcription, transient transfection assays were
performed with a promoter-luciferase reporter gene construct containing
IL-11 promoter elements between nucleotides
728 and +58 (relative to
the transcription start site). The levels of luciferase activity in
A549 cells were evaluated at base line and after TGF-
1
stimulation. As can be seen in Fig. 2,
only a modest level of luciferase activity was able to be detected in unstimulated A549 cells. In contrast, TGF-
1 was an
impressive, dose-dependent stimulator of the promoter
activity of this construct (Fig. 2). This demonstrates that the
728
to +58 fragment of the IL-11 promoter contains
TGF-
1-responsive sequences.
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Definition of the TGF-1 Response Element in the
IL-11 Promoter--
To define further the cis-element(s) in the IL-11
promoter that responds to TGF-
1, intrinsic restriction
sites and Bal-31 digestion were employed to obtain IL-11 promoter
fragments that contain progressively larger 5' deletions. These
promoter constructs were then cloned into our luciferase reporter
construct, and their responsiveness to TGF-
1 was
assessed. Constructs whose 5' end extended from
728 to
81 were
generated and tested. The parent (
728 to +58) and all of the 5'
deletion constructs did not express significant levels of luciferase
activity in unstimulated A549 cells. TGF-
1, however, was
a potent stimulator of IL-11 promoter-driven luciferase activity in the
parent construct (Fig. 3). Interestingly, 5' deletions extending from
728 to
100 did not significantly alter
TGF-
1 responsiveness (Fig. 3). In contrast, deletions
past
100 markedly diminished TGF-
1 responsiveness.
When stimulated with TGF-
1, these constructs had
5%
of the TGF-
1 inducibility of the wild type
728 to +58
parent construct (Fig. 3). These studies demonstrate that elements that
are essential for TGF-
1 induction exist proximal to
nucleotide
100 in the IL-11 promoter.
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Importance of AP-1 Sites in TGF--mediated Transcriptional
Activation--
Inspection of the sequence immediately 3' of
100 in
the IL-11 promoter demonstrated two AP-1-like elements separated by 3 base pairs (Fig. 4). To define the role
that these elements play in conferring TGF-
1
responsiveness, constructs were prepared that contained point mutations
at these sites, individually and in combination. Neither mutation
caused a significant increase in the basal levels of IL-11 promoter
activity (Fig. 5). Individual mutation in
the 5' (distal) or 3' (proximal) sites caused an approximately 75%
decrease in TGF-
1 responsiveness (Fig. 5).
Interestingly, the simultaneous mutation of both the 5' and the 3'
AP-1-like sites abrogated TGF-
1 responsiveness in this
system. These constructs had
5% of the TGF-
1
responsiveness of the wild type promoter-luciferase construct (Fig. 5).
These studies demonstrate that both of these AP-1 sites play important
roles in TGF-
1-induced IL-11 activation.
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Effect of TGF-1 on AP-1 Protein-DNA Binding--
To
gain additional insight into the trans-acting factors that bind to the
AP-1 sites in the IL-11 promoter, electrophoretic mobility shift assays
(EMSA) were performed using labeled oligonucleotides identical to the
5' and 3' AP-1-like sequences in the IL-11 promoter. At base line,
protein-DNA binding was detected at the 5' site but not the 3' site
(Fig. 6). TGF-
1
stimulation caused an impressive increase in binding to the 5' site.
This stimulation was noted 2-6 h and peaked approximately 12-24 h
after the addition of TGF-
1 to the A549 cell cultures
(Fig. 6). TGF-
1 stimulation also induced protein-DNA
binding at the 3' AP-1 site in the IL-11 promoter. This induction had a
slower kinetic than the induction at the 5' site. It was, however,
readily detected after 12-24 h of TGF-
1-cell incubation
(Fig. 6). In both cases, the protein-DNA binding was eliminated by the
addition of excess unlabeled oligonucleotides encoding the 5' AP-1
site, 3' AP-1 site, or a consensus AP-1 sequence but not by
oligonucleotides encoding NF-
B, AP-2, or SP-1 sequences (data not
shown). When viewed in combination, these studies demonstrate that
TGF-
1 enhances AP-1 family protein binding to both the
5' and 3' AP-1 sites in the IL-11 promoter.
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Effects of TGF-1 on AP-1 Subunit
Composition--
To understand further the trans-acting factors
binding to the AP-1 sites in the IL-11 promoter, supershift EMSAs were
performed using antibodies to a variety of AP-1 family proteins. At
both the 5' and 3' sites the importance of AP-1 family members was confirmed since anti-pan Jun and anti-pan Fos antibodies (antibodies against all Jun proteins and all Fos family proteins, respectively) caused impressive supershifts in this assay (Fig.
7 and data not shown). In addition,
characteristic patterns of AP-1 subunit usage were noted when selective
antibodies were employed. At base line, the binding to the 5' IL-11
AP-1 site was composed almost entirely of JunD AP-1 moieties (Fig. 7).
Interestingly, TGF-
1 increased the contribution of
c-Jun, Fra-1, and Fra-2 proteins while simultaneously decreasing the
contribution of JunD proteins to the 5' AP-1-DNA binding. Significant
alterations in c-Fos, FosB, and JunB were not noted (Fig. 7). Similar
alterations in AP-1 subunit binding to the 3' site were noted (data not
shown). These studies demonstrate that TGF-
1 stimulation
of IL-11 gene transcription is associated with impressive and selective
alterations in the composition of the AP-1 moieties that bind to the
IL-11 promoter. These changes are characterized by an enhanced
contribution from c-Jun, Fra-1, and Fra-2 at the 5' and 3' sites and a
simultaneous decrease in the base line binding of JunD to the 5'
site.
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TGF-1 Response Elements Confer Responsiveness on a
Minimal Promoter Construct--
To understand further the
TGF-
1 response elements in the IL-11 promoter, studies
were undertaken to determine if they could confer TGF-
1
responsiveness on a minimal promoter-reporter gene construct. This was
done by generating constructs containing IL-11 promoter fragments
between
103 and
77, in the sense and antisense direction, in series
with the herpes simplex virus minimal promoter and luciferase reporter
gene. The activity of these constructs in the presence and absence of
TGF-
1 was compared with the activity of the minimal
promoter-luciferase construct under the same conditions. The parent
minimal promoter-luciferase construct did not demonstrate significant
levels of activity in the presence or absence of TGF-
1 (Fig. 8). In contrast,
TGF-
1 was a potent stimulator of the sense and antisense
IL-11 promoter-minimal promoter-Luc constructs (Fig. 8). These studies
demonstrate that the sequences between
103 and
77 in the IL-11
promoter act as an enhancer and confer TGF-
1 responsiveness on heterologous promoter elements.
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DISCUSSION |
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Studies from a number of laboratories have demonstrated that
TGF-1 is a potent stimulator of IL-11 production. To
understand further the biology of IL-11, the mechanism of this
stimulation was characterized. Nuclear run-on studies demonstrated that
this stimulatory effect is, at least in part, transcriptionally
mediated, and transient transfection assays demonstrated that
TGF-
1 is a potent stimulator of IL-11 promoter-reporter
gene constructs. Two AP-1 motifs located between nucleotides
100 and
82 in the IL-11 promoter were noted to be necessary for this
induction. In addition, a 25-base pair nucleotide sequence that
contains these AP-1 sites was able to confer TGF-
1
responsiveness on a heterologous minimal promoter. Supershift EMSA
demonstrated that JunD is the major AP-1 protein binding promoter DNA
in unstimulated cells. TGF-
1 stimulation was associated
with enhanced protein-DNA binding to both AP-1 motifs with an augmented
contribution by c-Jun, Fra-1, and Fra-2 proteins and a decreased
contribution by JunD proteins. These are the first studies to
investigate, in depth, the mechanisms that stimulate IL-11 gene
transcription. All in all, they demonstrate the importance in this
inductive response of complex alterations in AP-1 transcription factors and a cis-enhancer element that contains two closely approximated AP-1
motifs.
TGF-1 is a pleiotropic cytokine with far reaching
effects on tissue homeostasis. Prominent in this regard are its ability to stimulate tissue fibrosis, regulate matrix molecule elaboration, and
inhibit tissue inflammation. In keeping with its biologic importance, a
significant amount of effort has been directed at characterizing the
mechanism(s) by which TGF-
mediates its biologic effects. Studies of
TGF-
1 regulation of cytokine production (33), cell
proliferation (34), and retinoic acid receptor expression (35) have
demonstrated that AP-1 activation can play a major role in these
processes. Our studies add to this body of data by demonstrating that
TGF-
1 stimulation of IL-11 elaboration is also mediated
via an AP-1-dependent pathway.
AP-1 was initially identified as a DNA binding activity in HeLa cell
extracts that bound to cis-elements within the promoter and enhancer
sequences of the human metallothionein IIA gene and simian virus 40 (36). It is now a term that is used to refer to dimeric proteins
produced by the complex immediate-early gene family that couple a
variety of extracellular stimuli from the cell surface to the nucleus
to initiate alterations in gene expression and cell phenotype (37, 38).
Investigations of these moieties have revealed a consensus binding site
for these dimers, the palindromic sequence, 5'-TGA(G/C)TCA-3' (36). Our
studies demonstrated that the sequence between 100 and
82 plays a
crucial role in the transcriptional activation of the IL-11 promoter.
Inspection of this region revealed a classic AP-1 binding site (the
distal (5') AP-1 sequence) and a binding site that differs at a single
nucleotide (the proximal (3') AP-1 sequence). Both sites were shown to
bind AP-1 moieties. In addition, mutation of each AP-1 site
significantly decreased and the simultaneous mutation of both AP-1
sites totally abrogated TGF-
1-induced IL-11 promoter
activation. Furthermore, oligonucleotides made up largely of these 2 AP-1 sites conferred TGF-
1 responsiveness upon a minimal
heterologous promoter. These studies demonstrate that both AP-1 sites
play important roles in IL-11 transcriptional activation and the
enhancer function of this crucial promoter region. This is in keeping
with studies of other genes such as involucrin (39) and tissue factor
(40) whose promoters contain multiple AP-1 sites that play important roles in gene activation.
TGF-1 is one of a number of stimuli that induce
epithelial cell IL-11 production in vitro. Previous studies
from our laboratory demonstrated that a variety of respiratory tropic
viruses also stimulate A549 cell production of IL-11 protein and the
accumulation of IL-11 mRNA (17, 19). The studies in this manuscript
demonstrate that RSV stimulates A549 cell IL-11 production via a
mechanism that is, at least in part, transcriptionally mediated. They
also demonstrate that the magnitude of induction of IL-11 induced by RSV is comparable to that seen with TGF-
1. At the start
of these studies, we hypothesized that RSV and TGF-
1
would activate the IL-11 promoter via similar mechanisms. This did not,
however, prove to be true. Although RSV was as potent as
TGF-
1 in the induction of IL-11 protein production,
mRNA accumulation, and gene transcription, it did not efficiently
stimulate the IL-11 promoter-reporter gene constructs used in these
studies. This demonstrates that TGF-
1 and RSV activate
the IL-11 promoter via different mechanisms. It also provides insight
into the stimulus specificity of the pathways used to activate IL-11
gene transcription.
AP-1 proteins are made up of homo- and heterodimers composed of Fos,
Jun, and activating transcription factor subunits (37). At least 24 different combinations have been described (36). This complexity is the
result of important tissue-specific, stimulus-specific, and temporally
regulated differences in AP-1 subunit expression (36, 40, 41). This
results in AP-1 moieties that differ in their DNA binding capacities,
transactivation capacities, and biologic effects (42-44). To gain
insight into the AP-1-mediated events involved in TGF-1
stimulation of IL-11, we used supershift gel mobility shift assays to
define the Fos and Jun proteins involved in this inductive response.
These studies demonstrate that, in the absence of stimulation, JunD is
the major AP-1 moiety that can be detected. This is in keeping with
studies from a number of different laboratories demonstrating that JunD
is constitutively expressed in a variety of cells and tissues (45) and
studies from our laboratories that demonstrate that JunD plays an
important role in the constitutive elaboration of IL-11 by PU34 cells
(21). Our studies also demonstrate that TGF-
1
stimulation is associated with an increase in c-Jun, Fra-1, and Fra-2
and a decrease in JunD-IL-11 promoter binding. These observations are
in accord with the known inducibility of these AP-1 subunits and the
well documented stimulus specificity of AP-1 subunit induction (36, 40,
43, 44, 46, 47). It is impossible to determine, from these studies, the
degree to which the TGF-
1-induced alterations in c-Jun,
Fra-1, Fra-2, and/or JunD individually contribute to the induction of
IL-11 studied in the present analysis. It is possible, however, to
hypothesize that all may play an important role. JunD is a potent
transactivator but, in contrast to many other AP-1 subunit moieties, is
not induced in a major fashion by extracellular stimuli (21, 45). The
JunD homodimers that would form in unstimulated cells under conditions
of JunD excess would therefore play an important role in the regulation
of basal IL-11 production but be unable to meet the enhanced
transcriptional demands after TGF-
1 stimulation. In
contrast, TGF-
1 stimulation enhances the contribution of
c-Jun, Fra-1, and Fra-2 resulting in AP-1 dimers that are responsive to
the conditions of stimulation. The entry of c-Jun, Fra-1, and Fra-2
into the AP-1·DNA binding complex can thus be speculated to be a key
event underlying TGF-
1-stimulated transcription of the
IL-11 gene in A549 cells. A similar transcriptional activation paradigm
has been proposed to explain the contribution of AP-1 in the
stimulation of tissue factor by serum in mouse fibroblasts (40).
Our studies demonstrate that two closely approximated AP-1 sites in the
IL-11 promoter play important roles in the stimulation of IL-11 gene
transcription by TGF-1. It is important to keep in mind,
however, that transcriptional activation is frequently a
multi-factorial process that involves the concerted and coordinated interaction of a number of different transcription factors. This is
well documented for the AP-1 moieties that are known to interact with a
variety of other transcription factors including nuclear factor-
B
(NF-
B), CREB, nuclear factor IL-6 (NF-IL-6), liver regeneration
factor-1 (LRF-1), and polyoma virus enhancer A binding protein-3
(PEA3) (36, 48). The transcriptional activities of AP-1
moieties are also regulated by a variety of other proteins including
the Jun dimerizing protein (37) and the Jun activation domain binding
protein 1 (JAB1) (37). In accord with this information, it is important
to point out that the present studies, while implicating AP-1 in the
regulation of TGF-
1-stimulated IL-11 transcription, do
not address the importance of each of these other moieties. It is
likely that additional investigations will demonstrate that other cis-
elements and/or trans-activating factors are involved in the
coordinated production of IL-11 under a variety of circumstances.
In summary, the present studies demonstrate that TGF-1
stimulates IL-11 gene transcription in A549 cells and that this
stimulation is mediated via a complex AP-1-dependent
activation pathway. They also highlight two closely approximated AP-1
sites in the IL-11 promoter that are essential for this activation and
demonstrate that DNA sequences that contain these two sites can confer
TGF-
1 responsiveness on a heterologous minimal promoter.
Lastly, these studies demonstrate that, in the absence of
TGF-
1 stimulation, JunD is the major AP-1 subunit
involved in IL-11 promoter-protein binding and that
TGF-
1 stimulation is associated with increased c-Jun,
Fra-1, and Fra-2 and decreased JunD-DNA binding.
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ACKNOWLEDGEMENTS |
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We acknowledge the scientists and institutions that provided the reagents that were employed. We thank Kathleen Bertier for excellent secretarial and administrative assistance and Drs. Anuradha Ray and Prabir Ray for their frequent helpful suggestions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL-36708, AI-34953, HL-54989, HL-56389 (to J. A. E.), DK-50570, and HL-48819 (to Y.-C. Y.).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: Section of Pulmonary and Critical Care Medicine, Yale University School of Medicine, Dept. of Internal Medicine, 333 Cedar St./105 LCI, New Haven, CT 06520-8057. Tel.: 203-785-4163; Fax: 203-785-3826; E-mail: jack.elias{at}yale.edu.
1
The abbreviations used are: IL, interleukin;
TGF-1, transforming growth factor-
1;
AP-1, activating protein-1; RSV, respiratory syncytial virus; DMEM,
Dulbecco's modified Eagle's medium; DTT, dithiothreitol; EMSA,
electrophoretic mobility shift assays; bp, base pair(s); tk, thymidine
kinase.
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REFERENCES |
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