From the Department of Research and Internal
Medicine, University Hospital, 4031 Basel, Switzerland and
The
Netherlands Cancer Institute, Division of Molecular Biology,
Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
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ABSTRACT |
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Transforming growth factor (TGF)- The transforming growth factor
(TGF)1- In mesenchymal derived cells such as fibroblasts, TGF- IL-6 expression is known to be affected by a variety of cytokines and
growth factors. Up-regulation of IL-6 is reportedly controlled by the
activity of several transcription factors with known consensus
sequences in the IL-6 promoter region, including AP-1, C/EBP- This study addresses the question how IL-6 expression is regulated by
TGF- Materials--
Unless otherwise specified, all reagents were
purchased from Sigma (Buchs, Switzerland). Cell culture media and
additives were purchased from Fakola/Seromed (Basel, Switzerland).
TGF- Cell Culture--
Primary human cell lines of fibroblasts
(n = 10) were established from human lung tissue
biopsies obtained from patients undergoing lobectomy or pneumectomy, as
described previously (18, 24). Tissues were cut in small slices, placed
in cell culture flasks precoated with fetal calf serum, and incubated
for 1 week in RPMI 1640 supplemented with 10% fetal calf serum, 8 mM L-glutamine, and 20 mM HEPES.
Fibroblasts had regularly grown out from the tissue slices after 1 week
and were passaged by standard trypsinization. For all experiments,
cells were used before their seventh passage. Fibroblasts were plated
onto 150-mm cell culture dishes until confluent. Cells were then
starved in low serum medium (0.1% fetal calf serum) for 24 h
prior to stimulation with TGF- RT-PCR--
Total RNA was extracted from TGF- Enzyme-linked Immunoassay--
Concentrations of IL-6 protein
secreted into culture supernatants of fibroblasts were determined by
enzyme-linked immunoassay (Amersham Pharmacia Biotech). Cells were
plated onto 24-well plates until confluent and starved in 0.1% fetal
calf serum for 24 h. Fibroblasts were stimulated with different
concentrations of TGF- Plasmid Construction, 5'-Deletion Constructs, and Site-directed
Mutagenesis--
A plasmid containing a 651-bp fragment of the human
IL-6 gene promoter located directly upstream of the transcriptional
start site was kindly provided by Shigeru Katamine (Nagasaki
University, Nagasaki, Japan) (25). The 651-bp insert was subcloned
(5'-KpnI, 3'-XhoI) into pGL3 basic luciferase
reporter gene vector to give the parental pIL6-luc651 construct. From
this parental construct, 5'-deletion mutants were made by deleting
fragments containing the consensus sequence for transcription factor
AP-1 (pIL6-luc220) or CREB (pIL6-luc160) using internal restriction
sites for NheI or AatII, respectively.
Within pIL6-luc651, the following consensus sites were inactivated by
site-directed mutagenesis. The AP-1 consensus sequence (positions Cell Transfection and Luciferase Assays--
Two days before
transfection, fibroblasts were seeded into 24-well plates (1 × 104 cells/well) precoated with 1% gelatin. After 24 h, cells were serum-deprived for 24 h and subjected to liposomal
transfection using the cationic lipid Tfx-50 at a DNA:lipid ratio of
1:3 (1 µg of plasmid/well) for 2 h. Cells were then overlaid
with low serum medium with or without TGF- Preparation of Cytosolic and Nuclear Extracts--
Nuclear and
cytosolic extracts from TGF- Electrophoretic Mobility Shift Assays--
DNA mobility shift
assays were performed using oligonucleotides comprising the
consensus sequences for AP-1 (5'-CGCTTGATGAGTCAGCCGGAA-3'), C/EBP- Statistical Analysis--
All data were obtained from at least
four different cell lines of primary human lung fibroblasts.
Enzyme-linked immunoassay and RT-PCR were performed in duplicate using
three independent sets of experiments. For statistical analysis,
Student's t test and analysis of variance were performed.
p < 0.01 was estimated significant.
Ethics Committee Approval--
The protocol for establishing
primary human cell cultures from biopsies obtained during lung surgery
was approved by the Ethics Committee of the School of Medicine,
University of Basel, Switzerland (approval number M75/97).
TGF-
RT-PCR, although not performed quantitatively, thus indicated that
TGF- TGF-
In order to identify cis-regulatory sequences that were
responsible for the up-regulation of IL-6 expression by TGF-
To further analyze this observation, we generated mutants of known
consensus sequences within the IL-6 promoter by site-directed mutagenesis (Fig. 2B). We tested the parental pIL6-luc651
with mutations in the AP-1 binding sequence, the C/EBP- AP-1 Is Specifically Activated by TGF- Characterization of the AP-1 Complex Induced by TGF-
We further analyzed the AP-1 complex by using antibodies specific for
distinct Jun isoforms (c-Jun, JunB, and JunD). As demonstrated in Fig.
4B, only the pan-c-Jun and the JunD antibody resulted in a
significant decrease of the specific AP-1 band, thus suggesting the
AP-1 complex to be a JunD homodimer. This was further confirmed by the
fact that only these two antibodies resulted in a clear supershifted
band. As a serum control, an antibody specific for the glucocorticoid
receptor (GR) supplied by the same manufacturer was used
(Fig. 4B). Since the addition of this antibody did not affect the specific band, unspecific effects of the antibodies used for
supershift analyses can be reasonably excluded. Furthermore, the
addition of a 50-fold excess of unlabeled competitor oligonucleotides diminished the appearance of the specific AP-1 band (Fig.
4B).
TGF- In this report, we investigated the molecular mechanisms of
TGF- AP-1 is composed as a homodimer of Jun isoforms or as a heterodimer of
Jun/Fos isoforms, both members of the immediate early gene family (31,
32). DNA binding of AP-1 occurs after activation and dimerization of
the described isoforms. Interestingly, the specificity of biological
effects induced by upstream stimuli is determined through the actual
composition of AP-1 in a certain signal transduction cascade. In this
respect, JunB-containing complexes are predominantly described to be
negative regulators of AP-1 function and inhibit transcription by
negative interaction with AP-1 consensus sequences (33). In contrast,
increased gene expression by AP-1 is mostly attributed to
c-Jun-containing complexes.
In our experiments, AP-1 was shown to up-regulate IL-6 expression, and
the composition of AP-1 was unequivocally determined by supershift
analyses with antibodies directed against distinct members of the
Jun/Fos family. These supershift analyses revealed that the
TGF- In an elegant study, Mauviel and colleagues have described how TGF- In addition to AP-1, Sp1 is another major transcription factor
frequently described as being activated by TGF- Besides AP-1 and Sp1, we investigated activation of C/EBP- Although these results imply that AP-1 is the immediate effector
molecule of TGF- CBP/p300, in contrast, are coactivators that need to bind to activating
transcription factors before potentiation of gene transcription can
occur (44). CBP is described to associate with AP-1 (45), thereby
leading to increased gene transcription that is controlled by AP-1
consensus sites within the promoter sequences investigated (44, 46). It
is therefore possible that TGF- Taken together, our findings thus underline the importance of AP-1 as a
major mediator of TGF-1 induces
extracellular matrix deposition and proliferation of mesenchymal cells.
We recently reported that interleukin (IL)-6 is an essential mediator
of growth factor-induced proliferation of lung fibroblasts. Here, we
demonstrate by reverse transcriptase polymerase chain reaction and
enzyme-linked immunoassay that TGF-
1 is a potent inducer of IL-6
mRNA and protein in primary human lung fibroblasts. Transient
transfections of fibroblasts with a luciferase reporter gene construct
containing nucleotides
651 to +1 of the human IL-6 promoter revealed
that TGF-
1 also potently activated IL-6 promoter activity.
Progressive 5'-deletions and site-directed mutagenesis of the parental
construct located the TGF-
1-responsive cis-regulatory
element to a known activating protein-1 (AP-1) sequence (nucleotides
284 to
276). Gel shift analyses revealed that AP-1 DNA binding
activity in nuclear extracts was increased 30 min after stimulation
with TGF-
1. In contrast, neither CCAAT enhancer-binding protein-
,
NF-
B, nor Sp1 were activated by TGF-
1. Supershift analyses
demonstrated that the AP-1 complex induced by TGF-
1 was composed of
Jun isoforms and absent of Fos isoforms. Moreover, this complex was
found to be a JunD homodimer. Our data thus demonstrate that TGF-
1
is a potent inducer of IL-6 in primary human lung fibroblasts. The TGF-
1-activated JunD homodimer may be essential for a majority of
the biological effects induced by TGF-
1 in this cell type, such as
proliferation and extracellular matrix synthesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
isoforms play an
essential role during organ development and tissue homeostasis.
Biological effects induced by TGF-
s are highly cell type- and
tissue-specific, depending on the state of differentiation of a given
cell type (1-4). The TGF-
superfamily consists of more than 20 members, most important of which are the TGF-
s themselves, activins,
and bone morphogenic proteins. Among the five described TGF-
isoforms, TGF-
1 is most abundant in human tissues (5, 6). Signal transduction of TGF-
1 requires ligand binding to TGF-
receptors (T
R). Three T
R have been characterized (T
RI to T
RIII),
T
RI and T
RII belong to the family of serine/threonine kinase
receptors. Upon ligand binding to T
RII, T
RII recruits T
RI to
form a heterooligomeric ligand-receptor complex. T
RI is then
phosphorylated at glycine and serine residues by the constitutively
active kinase T
RII, and downstream signaling is initiated (6, 7).
Studies investigating the cis- and trans-acting
elements of TGF-
-responsive genes have so far identified several
transcription factors directly involved in TGF-
signaling, such as
Sp1 (8, 9), AP-1 (10, 11), CTF/NF-1 (12), and SMAD proteins
(13-15).
s have two
major effects, (i) an increase in extracellular matrix
deposition and (ii) an increase in cellular proliferation
(16, 17). The proliferative response of fibroblasts to mitogenic
stimuli, such as TGF-
1, is preceded by increased DNA synthesis. In
this respect, we have previously shown that growth factor-induced
proliferation of lung tumor cell lines and primary human lung
fibroblasts is essentially mediated by interleukin (IL)-6 (18, 19).
Down-regulation of IL-6 levels by antisense treatment resulted in
abrogation of the proliferative response of fibroblasts to mitogenic
stimuli, whereas the response was not affected by antisense
oligonucleotides against other interleukins (18).
, and
NF-
B (20, 21). In a different cell culture model, TGF-
1 has been
reported to up-regulate IL-6 expression (22, 23); however, the
molecular mechanism of increased IL-6 expression in response to
TGF-
1 is unknown.
1 in primary human lung fibroblasts. We found that upon
stimulation with TGF-
1, IL-6 is rapidly up-regulated at the mRNA
and protein level. This genomic effect of TGF-
1 is reflected at the
promoter level, demonstrating rapid activation of AP-1, as shown by
luciferase and electrophoretic mobility shift assays. The
TGF-
1-induced AP-1 complex is found to be a JunD homodimer, while
Fos proteins are absent in this complex. Thus, our data provide new
insight into the signaling pathway induced by TGF-
1, a major growth
factor due to its prominent biological effects during induction of
fibrosis, in primary human lung fibroblasts.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 was from R & D Systems (Buckinghamshire, United Kingdom).
Antibodies to Jun and Fos isoforms were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Trizol reagent was purchased from
Life Technologies, Inc. (Basel, Switzerland). Premixed proteinase
inhibitors (CompleteTM) were from Roche Molecular
Biochemicals (Rotkreuz, Switzerland). [
-32P]ATP was
purchased from Amersham Pharmacia Biotech (Zürich, Switzerland).
PCR reagents were obtained from Perkin-Elmer. Agarose was purchased
from NuSieve GTG FMC BioProducts (Rockland, ME). Luciferase reporter
gene vector series pGL3 and pRL-TK, Luciferase assays, and Tfx-50
transfection reagents were purchased from Promega (Bethesda, MD). All
primers were synthesized by MWG-Biotech AG (Muenchenstein, Switzerland).
1 at the indicated concentrations. No
antibiotics or antimycotics were added to the culture conditions at any time.
1-stimulated or
control lung fibroblasts (3 × 107 cells) with Trizol
reagent as described (19). RNA was suspended in 30 µl of sterile
deionized water, and RNA concentrations were determined
spectrophotometrically at 260 nm. All RNA preparations had an
A260/A280 ratio of
>1.75. Aliquots of 1 µg of total RNA were transcribed into cDNA
at 42 °C for 15 min in a total volume of 20 µl containing 20 mM Tris HCI, pH 8.3, 50 mM KCl, 5 mM MgCl2, 1 mM of each dNTP, 1 unit
of RNase inhibitor, and 2.5 units of murine leukemia virus reverse
transcriptase. PCRs were then performed with 10 µl of the reverse
transcription reactions for amplification of IL-6 and
-actin
cDNA. Amplifications were performed in a total volume of 50 µl
containing 0.5 units of Taq polymerase and 15 pmol of
primers specific for IL-6 (5'-GCCCAGCTATGAACTCCTTCTC-3' and
5'-GAGTTGTCATGTCCTGCAGCC-3') or
-actin
(5'-GTACGTTGCTATCCAGGCTGTGC-3' and 5'-TCAGGCAGCTCGTAGCTCTTCTC-3').
Amplifications were performed with 25 cycles for
-actin and 35 cycles for IL-6 cDNA. The amplification profile included
denaturation at 98 °C for 15 s, primer annealing at 62 °C
for 15 s, and extension at 72 °C for 30 s, followed by a
final extension at 72 °C for 5 min. 12 µl of each PCR were
analyzed by agarose gel electrophoresis on 3.0% agarose gels.
1 as indicated, and aliquots of the
supernatants were removed at several time points. Measurements of IL-6
were carried out according to the manufacturer's instructions.
283
to
276, 5'-TGAGTCAC-3') was changed to 5'-TGCAGCAC-3'; the C/EBP-
consensus sequence (positions
154 to
146, 5'-TTGCACAAT-3') was
changed to 5'-CCGTTCAAT-3'; and the NF-
B consensus sequence (positions
72 to
63, 5'-GGGATTTTCC-3') was changed to
5'-CTCATTTTCC-3'. These mutations have previously been shown to
inactivate the described consensus sequences (26, 27). All mutant
clones were verified by DNA sequencing.
1 at the indicated
concentrations. After 36 h of incubation, cells were harvested by
active lysis, and equal amounts of lysates were analyzed for firefly
luciferase expression according to the directions of the manufacturer.
In brief, 20-µl aliquots of cell lysates were mixed with 100 µl of luciferase reagent buffer, and luminescence of the samples was integrated over a time period of 10 s in a LUMAC Biocounter M1500P (Landgraaf, The Netherlands). As an internal control for transfection efficiency, expression plasmids encoding Renilla luciferase
driven by the thymidine kinase promoter were used (0.4 µg/well).
Firefly and Renilla Luciferase have distinct substrate
properties, and thus activity of both enzymes can be assessed in the
same sample using two substrates sequentially (Dual Luciferase Assay, Promega).
1-stimulated or -unstimulated control
cells were prepared for gel shift analyses at the indicated time
points. Cells were washed twice in ice-cold phosphate-buffered saline
and harvested in 1 ml of phosphate-buffered saline with a rubber
policeman. Samples were centrifuged for 1 min at 6,000 × g (4 °C), and the resulting cell pellets were resuspended in 100 µl of low salt buffer (20 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM NaVO4, 1 mM EDTA, 1 mM EGTA, 0.2% Nonidet P-40, 10%
glycerol, supplemented with a set of proteinase inhibitors,
CompleteTM). After 10 min of incubation on ice, the samples
were centrifuged at 13,000 × g for 2 min (4 °C),
and the supernatants (cytosolic extracts) were immediately frozen in a
dry ice/ethanol bath. Pelleted nuclei were resuspended in 60 µl of
high salt buffer (20 mM Hepes, pH 7.9, 420 mM
NaCl, 10 mM KCl, 0.1 mM NaVO4, 1 mM EDTA, 1 mM EGTA, 20% glycerol, supplemented
with CompleteTM), and nuclear proteins were extracted by
shaking on ice for 30 min. Samples were centrifuged at 13,000 × g for 10 min (4 °C), and the supernatants were taken as
nuclear extracts.
(5'-GTAAGATTGCACAATGTA-3'), NF-
B
(5'-AGTTGAGGGGATTTCCCAGGC-3'), or Sp1
(5'-ATTCGATCGGGGCGGGCGAGC-3'). Oligonucleotides were end-labeled with [
-32P]ATP using T4-polynucleotide-kinase.
Aliquots of nuclear extracts (2 µg) were incubated with the labeled
consensus oligonucleotides under binding conditions (4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 50 µg/ml poly(dI-dC)) in a total
volume of 10 µl. Incubations were carried out at room temperature for
30 min, and protein-DNA complexes were analyzed on a 4% polyacrylamide
gel. The identity of specific bands was confirmed by the addition of
competitive unlabeled consensus sequence oligonucleotides and by the
addition of monoclonal antibodies specific for the transcription factor
of interest.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 Increases IL-6 mRNA and Protein Synthesis in Primary
Human Lung Fibroblasts--
We analyzed whether human lung
fibroblasts, in culture, up-regulate IL-6 expression in response to
TGF-
1 by qualitative RT-PCR. Fig.
1A demonstrates a
characteristic agarose gel of RT-PCR products amplifying IL-6 and
-actin mRNA from cells treated with TGF-
1 at different
concentrations for 8 h. The expected size of IL-6 mRNA was 565 bp, and the expected size of the constitutive message for
-actin
serving as an internal control was 225 bp. RT-PCR indicated that human
lung fibroblasts up-regulated IL-6 mRNA in response to TGF-
1
(0.01-1.0 ng/ml) in a concentration-dependent manner (Fig.
1A). As shown in Fig. 1A, IL-6 mRNA was
clearly up-regulated as compared with control levels 8 h after
stimulation with TGF-
1.
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Fig. 1.
Effect of TGF- 1 on
the induction of IL-6 mRNA and protein levels. A,
effect of TGF-
1 on IL-6 mRNA levels in quiescent primary human
lung fibroblasts. Confluent fibroblasts were treated with the indicated
concentrations of TGF-
1 for 8 h, and total RNA was isolated and
analyzed by qualitative RT-PCR. RT-PCR was performed with primers
specific for IL-6 (yielding an amplified product of 565 bp), as well as
for
-actin (yielding a product of 225 bp) as an internal control.
The gels shown are representative of four independent experiments using
different fibroblast cultures. B, effect of TGF-
1 on IL-6
protein secretion into culture supernatants of quiescent fibroblasts.
Supernatants of fibroblasts were collected 12 h after treatment
with TGF-
1 at the indicated concentrations and analyzed for the
amount of IL-6 by enzyme-linked immunoassay. Data shown are derived
from three independent measurements done in duplicate from one cell
line. Similar data were obtained using three different fibroblast
lines.
1 led to a dose-dependent increase in IL-6 message. We therefore analyzed whether the increased IL-6 mRNA levels
coincided with enhanced IL-6 secretion. IL-6 protein levels in culture
supernatants of lung fibroblasts treated with TGF-
1 for 12 h
were measured by enzyme-linked immunoassay. As shown in Fig.
1B, the levels of IL-6 protein were similarly increased in a
concentration-dependent manner as compared with controls.
Unstimulated quiescent human lung fibroblasts secreted 260 ± 13 pg/ml of IL-6 protein within 12 h. IL-6 secretion increased to
886 ± 23 pg/ml in response to TGF-
1 at a concentration of 1 ng/ml (p < 0.001), corresponding to a 341% increase.
When lung fibroblasts were stimulated with TGF-
1 at concentrations
of 0.1 or 0.01 ng/ml, IL-6 protein concentrations in culture
supernatants increased to 723 ± 43 pg/ml (278% increase) and
435 ± 23 pg/ml (167% increase), respectively (p < 0.001) (Fig. 1B).
1 Transactivates the Human IL-6 Promoter via an
AP-1-responsive Element--
We analyzed possible mechanisms
underlying IL-6 up-regulation by TGF-
1 using luciferase reporter
gene assays. We subcloned a 651-bp fragment of the human IL-6 promoter
into a luciferase reporter gene vector to give the parental construct
pIL6-luc651. As shown in Fig.
2A, TGF-
1 (1 ng/ml)
activated the parental pIL6-luc651 construct to 220 ± 12% of
unstimulated controls (1162 ± 98 relative light units (RLU)
versus 528 ± 25 RLU), when transiently transfected into human lung fibroblasts. Using TGF-
1 at a concentration of 0.1 ng/ml, IL-6 promoter activity was still increased by 258 ± 15%
(1061 ± 85 versus 528 ± 25 RLU) (Fig.
2A). Inducibility of the IL-6 promoter decreased to 144 ± 11% when TGF-
1 was used at 0.01 ng/ml (760 ± 62 versus 528 ± 25 RLU) (Fig. 2A), thus
reflecting the dose dependence observed on the mRNA and protein
level.
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Fig. 2.
Effect of TGF- 1 on
human IL-6 promoter transcriptional activity. A, effect
of TGF-
1 on promoter activity of the parental pIL6-luc651 construct
spanning 651 bp directly upstream of the transcriptional start site of
the IL-6 gene (see B). Fibroblasts were plated into 24-well
plates at a density of 5 × 104 cells/well and
cotransfected with pIL6-luc651 and pRL-TK (encoding Renilla
luciferase driven by the thymidine kinase promoter as an internal
control) by lipofection for 2 h. The DNA/lipid ratio used for all
experiments was 1:3. Transfected cells were stimulated with the
indicated amounts of TGF-
1, harvested after 24 h, and processed
for luciferase assays. RLU representing luciferase activity were
measured over 10 s in a luminometer and corrected for transfection
efficiency using the readout for Renilla luciferase. Data
represented were obtained from four independent experiments and are
representative for four different cell lines. B, deletions
of the parental pIL6-luc651, as well as mutants for the indicated
consensus sites, were generated as described under "Experimental
Procedures." Constructs were transiently transfected into human lung
fibroblasts along with the pRL-TK as described above. Induction of
promoter activity by TGF-
1 was assessed for the respective
constructs, corrected for transfection efficiency, and calculated as
percentage of induction compared with unstimulated base-line activity.
Measurements were done in triplicate and are representative of four
different cell lines. CRE, cAMP response element.
1, we generated several deletion mutants of the parental pIL6-luc651 construct. Two deletion mutants were screened for their TGF-
1 inducibility and compared with the parental pIL6-luc651: (i)
pIL6-luc220, deficient in a known AP-1 consensus sequence and (ii)
pIL6-luc160, deficient in both a known AP-1 and a known CREB consensus
sequence (see schematic diagram in Fig.
2B). As demonstrated in Fig. 2B, IL-6 promoter
inducibility by TGF-
1 was only observed with the parental
pIL6-luc651, suggesting that the TGF-
1-responsive
cis-regulatory sequences are located between nucleotides
651 to
220 of the IL-6 promoter.
binding
sequence, and the NF-
B binding sequence as well as a double mutant
of the C/EBP-
and NF-
B sequence for their TGF-
1 inducibility.
Interestingly, only the AP-1 mutant was found to lack TGF-
1
inducibility compared with the parental pIL6-luc651 (Fig.
2B). Neither the C/EBP-
, the NF-
B, nor the double
mutant demonstrated loss of TGF-
1 inducibility as compared with the
parental pIL6-luc651. Thus, the results obtained from luciferase assays
strongly suggest that AP-1 is specifically required for
TGF-
1-induced IL-6 gene expression in primary human lung fibroblasts.
1--
We assessed
whether AP-1 is specifically activated by TGF-
1 by performing
electrophoretic mobility shift analyses (EMSA) with nuclear extracts of
TGF-
1-treated and -untreated control fibroblasts. Time course
analyses with
-32P-labeled oligonucleotides spanning the
consensus sequences of transcription factors important for IL-6
promoter activity were performed. Fig. 3
includes characteristic EMSA demonstrating time course analyses of the
transcription factors AP-1 (Fig. 3A), C/EBP-
(NF-IL-6)
(Fig. 3B), and NF-
B (Fig. 3C) in nuclear
extracts of fibroblasts treated with TGF-
1 at 2 ng/ml. Over a
representative time course, only AP-1 DNA binding activity in nuclear
extracts of TGF-
1-treated fibroblasts was significantly increased
(Fig. 3A); none of the aforementioned transcription factors
were activated (Fig. 3, B and C). AP-1 activation
increased as early as 30 min after stimulation with TGF-
1, peaked at
5 h, and returned to base-line levels over a time frame of 16 h (Fig. 3A). Both C/EBP-
(Fig. 3B) and NF-
B
(Fig. 3C) exhibited constitutive DNA binding in nuclear
extracts of quiescent lung fibroblasts. However, in contrast to AP-1,
neither of the two was affected by TGF-
1 treatment. To further
demonstrate specificity of AP-1 activation, we also performed EMSA
investigating the transcription factors Sp1 and CREB. Although Sp1 is
reported to be activated by TGF-
1 in different cell types, none of
these two transcription factors were induced by TGF-
1 in human lung
fibroblasts (data not shown).
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Fig. 3.
EMSA characterizing nuclear protein binding
to AP-1, C/EBP- , and
NF-
B consensus site oligonucleotides.
Human lung fibroblasts were treated with medium alone or TGF-
1 at 2 ng/ml for the indicated times, and nuclear proteins were extracted as
described under "Experimental Procedures." Total protein was
calculated by Bradford assay, and equal amounts of protein (2 µg)
were used for EMSA. A, characteristic EMSA that demonstrates
increased binding of nuclear proteins from TGF-
1-treated fibroblasts
to an AP-1 consensus site-containing oligonucleotide. The specific AP-1
band is indicated on the right, as is an unspecific band
representing constitutive binding of nuclear proteins to the
oligonucleotide. Free probe is shown at the bottom of the
gel. B, the same extracts were prepared for binding to a
C/EBP-
oligonucleotide. On the left side, the labeled
oligonucleotide alone without cell extract and a positive control using
nuclear extracts from HeLa cells are shown. Afterward, supershift analyses of the
HeLa extract using an antibody specific for C/EBP-
(NF-IL6) and
competition analyses with a 50-fold excess of unlabeled probe are
shown. Analyses of nuclear extracts of TGF-
1-treated lung
fibroblasts (from 0 to 16 h) revealed no change in C/EBP-
(NF-IL6) binding. C, EMSA demonstrating constitutive NF-
B
binding activity in nuclear extracts of lung fibroblast, irrespective
of treatment with TGF-
1. The left part of Fig.
3C demonstrates binding activity of NF-
B in nuclear
extracts of human lung fibroblasts that is not affected by TGF-
1
over a time course of 16 h. The far left
lane represents unstimulated extract (0 h) with the addition
of a 50-fold excess of unlabeled oligonucleotide. The right
part of Fig. 3C is a supershift analyses of
NF-
B activity in control synovial fibroblasts known for
TNF-
-induced NF-
B activation. Specific NF-
B binding activity
is indicated on the right as well as unspecific binding of
the extracts to the oligonucleotide. Supershift analyses are performed
with extracts of TNF-
-treated synovial fibroblasts using a
p65-specific antibody at the indicated titers. The far
right lane represents TNF-
-stimulated extract
with the addition of a 50-fold excess of unlabeled
oligonucleotide.
1--
AP-1
is a dimeric transcription factor that either consists of Jun
homodimers or Jun/Fos heterodimers. We therefore analyzed the
composition of the TGF-
1-induced AP-1 complex by supershift analyses
with the addition of antibodies recognizing all Jun isoforms (pan-c-Jun/AP-1) or all Fos isoforms (pan-c-Fos). As demonstrated in
Fig. 4A, the antibody
recognizing all Jun isoforms clearly diminished the appearance of the
induced AP-1 complex, whereas the unspecific band was unaffected. In
contrast, the antibody specific for all Fos isoforms did not cause a
reduction of the specific AP-1 band, thereby suggesting the absence of
Fos isoforms in this specific AP-1 complex in human lung
fibroblasts.
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Fig. 4.
Supershift analyses of the
TGF- 1-induced AP-1 complex. Human lung
fibroblasts were treated with 2 ng/ml TGF-
1 for 5 h, and
nuclear extracts were prepared as outlined before. 2 µg of nuclear
proteins were incubated with an AP-1 oligonucleotide and analyzed by
supershift analyses. A, antibodies reactive against all
isoforms of either Jun or Fos, as indicated, were added to the binding
conditions at a titer of 1:10. Binding of nuclear proteins was then
analyzed by EMSA. The appearance of the specific AP-1 band is indicated
on the right. EMSA is representative for six independent
experiments with nuclear extracts from six different fibroblast lines.
B, the AP-1 complex was further analyzed by the addition of
Jun isoform-specific antibodies, the addition of a glucocorticoid
receptor (GR) antibody from the same supplier as a serum
control, and the addition of a 50-fold excess of unlabeled
oligonucleotide, as indicated. A specific band representing bound AP-1
complex is indicated on the right, as well as supershifted
bands and unspecific binding of nuclear proteins. The EMSA shown is
representative for six experiments using nuclear extracts of different
fibroblast lines.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
s play an essential role in the pathophysiology of lung
fibrosis. They have been shown to be causally responsible for the
changes associated with the fibroproliferative response in fibrotic
lungs, such as fibroblast proliferation and increased extracellular
matrix synthesis (28, 29). Studies investigating signal transduction by
TGF-
s have thus far revealed several transcription factors that are
activated upon stimulation with TGF-
s in a tissue- and cell
type-specific manner (2, 30), but although human lung fibroblasts are
highly responsive to TGF-
s, signal transduction pathways activated
by TGF-
in this cell type remain to be characterized.
1-induced IL-6 expression, a target gene known to be involved in
fibroblast proliferation (18, 19). Using cultures of primary human lung
fibroblasts derived from different patients undergoing pneumectomy/lobectomy, we demonstrate that TGF-
1 is a potent inducer
of IL-6 mRNA and protein expression in this cell type. Luciferase
and electrophoretic mobility shift assays demonstrate that the
transcription factor AP-1 is required for the observed induction of
IL-6 by TGF-
1. In human lung fibroblasts, none of the other
transcription factors that have previously been reported to be
activated by TGF-
s, such as Sp1, were affected in their DNA binding
activity. Thus, these results strongly indicate that, in addition to
its effect on IL-6 expression, AP-1 is a major signal transducer of
TGF-
1 and is required for most of its biological effects in human
lung fibroblasts. This notion is further strengthened by the fact that
all results were obtained with fibroblast cultures derived from
multiple patients, thus excluding genotype-specific effects and
implicating a general role for AP-1 in the human lung.
1-induced AP-1 complex exclusively consisted of JunD homodimers,
which have thus far not been described to be a dominant part of AP-1 in
TGF-
signaling. Although several investigations have found AP-1
activation upon TGF-
treatment in different cell types (30, 34, 35),
supershift analyses have revealed that, for the most part, c-Jun
isoforms constituted these AP-1 complexes. Consistent with our results,
Fos isoforms were generally not affected by TGF-
.
induces JunB in dermal fibroblasts, leading to decreased expression of
the matrix metalloproteinase-1 gene (36). In the same study, the
authors found that TGF-
up-regulated matrix metalloproteinase-1 expression in epidermal keratinocytes. In this cell type, the increase
in matrix metalloproteinase-1 expression was mediated by c-Jun, thereby
emphasizing the importance of different Jun isoforms in cell
type-specific effects of TGF-
. Investigation of a different target
gene, the matrix metalloproteinase-3 gene, revealed that up-regulation
of this metalloproteinase by TGF-
was due to an AP-1 complex that
consisted of c-Fos, c-Jun, and JunD (37). Moreover, AP-1 has been found
to transduce gene induction by TGF-
s in the cases of
2(I)
collagen (10), clusterin (38), plasminogen activator inhibitor-1 (39),
retinoic acid receptors (35), and TGF-
1 (11) itself.
s, as in the case of
1(I) collagen (40) and p21 gene expression (41). We therefore
determined the effects of TGF-
1 on Sp1 in human lung fibroblasts to
assess specificity and to generate a more complete analysis of TGF-
1
signaling in this cell type. Although fetal human skin fibroblasts have
been reported to activate Sp1 in response to TGF-
1 (40), we did not
detect any up-regulation of Sp1 in adult lung fibroblasts (data not
shown). These different observations, however, may result (i) from the
different tissue compartments from which fibroblasts were generated
(skin versus lung) or (ii) from the fact that fetal
fibroblasts react developmentally distinct from adult fibroblasts.
, CREB,
and NF-
B in this study, transcription factors known to be important
in the regulation of IL-6 gene expression (20, 27). None of these
factors were affected by TGF-
1, and mutations of their respective
consensus sequences within the IL-6 promoter did not affect IL-6 gene
induction by TGF-
1. In our model, AP-1 was the only transcription
factor of all those investigated found to be activated by TGF-
1 in
adult human lung fibroblasts.
1 signaling, we cannot exclude the possibility that
other factors contribute to AP-1-driven gene expression in this model.
In this respect, CBP/p300 or SMADs are of significant interest. In
recent years, SMAD proteins have evolved as essential elements in
TGF-
signaling (2, 13, 14). They become phosphorylated and activated
by ligand-bound T
R family members, associate in specific
heterodimers, and translocate to the nucleus, where they control gene
transcription. Interestingly, control of gene expression by SMADs is
achieved either by association with already DNA-bound factors or by
direct interaction with cis-regulatory elements (13, 15, 16,
42, 43). SMADs have been found to potentiate AP-1-dependent
gene transcription, but their precise nuclear function in this context
remains unknown (15, 16). Thus, the contribution of SMADs in the
described TGF-
1-induced transcriptional activation by AP-1 needs to
be fully resolved.
1 may increase CBP activity or its
association with AP-1, which would also lead to an increase in
AP-1-driven gene transcription, as observed for IL-6 expression in the
above described studies.
1 signaling in human lung fibroblasts. This
can also apply to physiological and pathophysiological conditions in
the lung in vivo, such as in diseases evoked by increased
TGF-
expression. In lung fibrosis, fibroblasts are known to be the key cell type responsible for the transition toward a fibrotic extracellular matrix. In this disease, AP-1 inhibition with special regard to JunD might thus be of crucial importance toward the development of novel therapeutic strategies.
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ACKNOWLEDGEMENTS |
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We thank Drs. M. Centrella and T. McCarthy for stimulating discussions.
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FOOTNOTES |
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* 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.
This work is dedicated to the memory of J. Eickelberg, father of O. E., who unexpectedly died while this manuscript was being reviewed.
§ A Feodor-Lynen-Fellow supported by the Alexander von Humboldt-Association. To whom correspondence should be addressed: Dept. of Pathology, Yale University School of Medicine, 310 Cedar St., LB 08, New Haven, CT 06520-8023. Tel.: 203-785-3090; Fax: 203-785-3348; E-mail: oliver.eickelberg{at}yale.edu.
¶ The first two authors contributed equally to this work.
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ABBREVIATIONS |
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The abbreviations used are:
TGF, transforming
growth factor;
IL, interleukin;
AP-1, activating protein-1;
TR, TGF-
receptor, EMSA, electrophoretic mobility shift assay;
bp, base pair(s);
PCR, polymerase chain reaction;
RT-PCR, reverse
transcriptase-PCR;
RLU, relative light units;
C/EBP, CCAAT
enhancer-binding protein;
CREB, cAMP response element-binding
protein.
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