(Received for publication, September 12, 1995; and in revised form, November 27, 1995)
From the
Previous studies have shown that transforming growth
factor- (TGF-
) and tumor necrosis factor-
(TNF-
)
modulate type I collagen gene expression in fibroblasts. To fine-map
the corresponding response elements in the human
2(I) collagen
(COL1A2) promoter, we have generated a series of 5` deletion
promoter/chloramphenicol acetyltransferase (CAT) reporter gene
constructs. Transient cell transfection assays using human dermal
fibroblasts and stable transfection experiments using NIH 3T3
fibroblasts identified the region located between residues -265
and -241, as critical for TGF-
response. Specifically, we
demonstrate that this 25-base pair region mediates the up-regulatory
effect of TGF-
on COL1A2 promoter activity and allows antagonistic
activity of TNF-
on the TGF-
effect. Gel mobility shift
assays indicate that nuclear factor binding to this 25-base pair region
of COL1A2 promoter is competed by AP-1, but not NF-1 or NF-
B,
oligonucleotides. Transient cell transfection experiments with plasmid
constructs in which the potential AP-1-binding site located within this
short region of promoter was modified by sitedirected mutagenesis
indicated that this element plays a significant role in the basal
activity of the promoter. Furthermore, this sequence is essential for
TGF-
response and does not require the presence of the three
Sp-1-binding sites located further upstream, between nucleotides
-273 and -304. In addition, overexpression of c-jun in co-transfection experiments with COL1A2 promoter/CAT constructs
blocks the TGF-
response, further implicating AP-1 in the
regulation of COL1A2 gene expression. Our results clarify the molecular
mechanisms involved in the regulation of type I collagen gene
expression and further emphasize the importance of AP-1 in mediating
some of the TGF-
effects on gene transcription.
Recently, significant progress has been made in understanding
the expression of the human 2(I) collagen (COL1A2) gene and its
transcriptional regulation by cytokines and growth factors. In
particular, it has been shown that a GC-rich region located between
residues -303 and -271, containing Sp-1-binding sites, is
important for high basal promoter activity (Tamaki et al.,
1995). This region is comprised within a larger segment of the COL1A2
promoter which has been shown to confer both TGF-
(
)(Inagaki et al., 1994) and TNF-
responsiveness (Inagaki et al., 1995). However, despite
extensive analyses, these studies did not allow precise
characterization of TGF-
or TNF-
-response element(s) within
the COL1A2 promoter. It was suggested that the TGF-
-responsive
element (TbRE) is located within a 131-bp region, between nucleotides
-378 and -255, and consists of at least two cis-elements which act in a concerted manner to mediate the
effect of TGF-
. Once inserted upstream of the thymidine kinase
promoter, the TbRE confers TGF-
inducibility to this heterologous
promoter. Two protein binding sequences within the TbRE, box 3A between
residues -313 and -286 which contains Sp-1 binding sites,
and box B between residues -271 and -255, were shown to
interact to confer both nuclear protein binding and promoter
inducibility, otherwise not observed with either box alone. In
addition, these authors suggested that TNF-
inhibitory effect
requires the contribution of both the Sp-1 binding sequence of the TbRE
and an inhibitory element, box 5A, immediately upstream of the TbRE,
but excluded, using an antibody interference experiment, participation
of both AP-1 and NF-
B transcription factors in this phenomenon
(Inagaki et al., 1995).
To characterize the TGF- and
TNF-
response elements within the human COL1A2 promoter in further
detail, our experimental approach consisted of (a) development
of a repertoire of 5` deletion constructs of the COL1A2 promoter and (b) site-directed mutagenesis of specific sequences
characterized as essential for growth factor response. This approach
allowed us to map the growth factor response elements, in the presence
of homologous downstream sequences, reaching the position +58 of
the COL1A2 gene. Specifically, we have narrowed the TGF-
response
element(s) to a 25-bp segment of the promoter, located between residues
-265 and -241. In addition, we show that this fragment is
sufficient to allow inhibition of the promoter activity by TNF-
.
Furthermore, using site-directed mutagenesis, we have established that
the potential AP-1-binding site, CGAGTCA, located within this short
region of promoter, is essential for TGF-
response.
Figure 3:
Binding of nuclear proteins to a DNA
fragment spanning the region from -271 to -235 of the
COL1A2 promoter. A 37-bp oligonucleotide including the sequence found
between residues -271 and -235 of the human COL1A2 promoter
was end labeled with [-
P]ATP and used in
gel electrophoresis mobility shift assays to study its ability to bind
transcription factors. A, the nuclear extracts used were as
follows: lane 1, none; lanes 2 and 4-7, untreated confluent fibroblast control cultures; lane 3, TGF-
-treated confluent fibroblast cultures.
Competition binding studies were performed as follows: lanes
1-3, no competitor DNA; lanes 4 and 5, 20-
and 60-fold molar excess of homologous unlabeled oligomer; lane
6, 20-fold molar excess of a 15-bp oligonucleotide corresponding
to the region -258 to -244 of the COL1A2 promoter; lane
7, 20-fold molar excess of a 22-bp oligonucleotide containing the
collagenase AP-1-binding site (underlined),
5`-CTAGTGATGAGTCAGCCGGATC-3`. B, the nuclear extracts used
were as follows: lane 1, none; lanes 2 and 4-9, untreated confluent fibroblast control cultures; lane 3, TGF-
-treated confluent fibroblast cultures.
Competition binding studies were performed as follows: lanes
1-3, no competitor DNA; lanes 4 and 5, 20-
and 60-fold molar excess of homologous unlabeled oligonucleotide; lanes 6 and 7, 20- and 60-fold molar excess of a
26-bp oligonucleotide containing the mouse COL1A2 NF-1 binding sequence
(underlined), 5`-CTTCCAAACTTGGCAAGGGCGAGAGA-3` (Rossi et al.,
1988); lanes 8 and 9, 20- and 60-fold molar excess of
a 22-bp oligonucleotide containing a consensus NF-
B binding
sequence (underlined) found in the immunoglobulin
light chain
gene, 5`-GATCGAGGGGACTTTCCCTAGC-3` (Lenardo and Baltimore,
1989).
Figure 1:
5` deletion analysis of the
human COL1A2 promoter. A, schematic representation of the
human COL1A2 promoter indicating the positions of the 5` ends of the
promoter/CAT reporter gene constructs used in transient cell
transfection experiments. The three potential Sp-1 as well as the
potential AP-1 and NF-kB binding sites are depicted as boxes,
and their respective nucleotide sequences are shown. B,
functional activity of the various 5` end deletion constructs in
transient cell transfection experiments of human neonatal skin
fibroblasts. Confluent fibroblast cultures were co-transfected with the
5` deletion constructs of the COL1A2 promoter linked to the CAT gene,
together with pRSV--galactosidase, by the calcium phosphate/DNA
co-precipitation procedure, as described under ``Materials and
Methods.'' After glycerol shock, the cultures were incubated in
medium containing 1% fetal calf serum for another 40 h prior to CAT
assay. The cell extracts corresponding to the same levels of
-galactosidase activity were assayed for CAT activity using
[
C]chloramphenicol as a substrate by thin layer
chromatography. The results are the mean of fifteen independent
experiments utilizing overlapping sets of deletion constructs. The
position of the 5` end of each construct is indicated below each histogram bar and is aligned with the schematic diagram shown
in A.
Additional deletions to position
-241 and to position -161, which removed two potential
regulatory elements, AP-1- and NF-B-binding sites (see Fig. 1A), did not lead to further reduction in promoter
activity (Fig. 1B), suggesting that the elements
important for the basal expression of the COL1A2 promoter are mostly
the Sp-1 sites described above and the sequences upstream of nucleotide
-772. Further deletion to position -108 reduced the
promoter activity to levels about 2-5% of the 3.5-kilobase
promoter construct, suggesting that sequences between -161 and
-108 are a prerequisite for the expression of COL1A2 at a
significant level.
Figure 2:
Effects of TGF- and TNF-
on the
activity of 5` deletions of the COL1A2 promoter in transient cell
transfection experiments. Confluent fibroblast cultures were
transfected with various 5` deletion constructs of the human COL1A2
promoter linked to the CAT gene by the calcium phosphate/DNA
co-precipitation procedure, as described under ``Materials and
Methods.'' After glycerol shock, the cultures were first incubated
for 3 h in medium containing 1% fetal calf serum and then for another
40 h with either TGF-
(10 ng/ml, B) or TNF-
(25
ng/ml, C). In every experiment, duplicate culture plates were
used for each case. The cell extracts were assayed for CAT activity
with [
C]chloramphenicol as a substrate using
identical amounts of protein. Results are the mean ± S.D. of 13
(TGF-
) and 10 (TNF-
) experiments utilizing overlapping sets
of 5` deletion constructs. Panel A allows visual
identification of the potential cis-elements involved in
growth factor response with regard to the 5` deletions of the COL1A2
promoter.
To verify the TGF- responsiveness in a
stable expression system, NIH 3T3 fibroblast cultures stably
transfected with constructs -342, -287, -265, and
-241/CAT were generated. Incubation of these transfectants with
TGF-
confirmed the data obtained in transient transfections of
human dermal fibroblasts, indicating that the segment of promoter
located between residues -265 and -241 is indeed essential
for TGF-
response. Specifically, all stably transfected
constructs, except -241/CAT, responded to TGF-
by a
3-5-fold elevation of promoter activity (not shown). These data
contrast those by Inagaki et al.(1994) which suggested that
sequences extended to span nucleotides -330 to -286 had to
be present, together with the sequences located between residues
-271 and -255, to confer TGF-
responsiveness to a
heterologous promoter, the thymidine kinase promoter, normally
unresponsive to TGF-
. The experimental approach taken by these
authors consisted in analyzing the region of the COL1A2 promoter
located between residues -378 and -183 by DNase
footprinting. Two distinct areas protected from nuclease digestion were
characterized, one between nucleotides -271 and -255, the
other between nucleotides -330 and -286. Deletion of either
one of the protected fragments led to significant reduction in the
promoter activity, suggesting that these two regions of COL1A2 promoter
are necessary for TGF-
effect. In our experiments, upstream Sp-1
sequences were found to be fundamental for high expression of the
promoter, as demonstrated by Tamaki et al.(1995), but are not
required for TGF-
response. Further evidence for the lack of
involvement of Sp-1 in TGF-
response is provided below (see Fig. 3and Fig. 5and the corresponding text).
Figure 5:
Effect of a mutation in the AP-1-binding
site on COL1A2 promoter activity and its regulation by growth factors.
Two 5` deletion constructs (-342 and -265) were subjected
to substitution mutations in the potential AP-1-binding site located at
position -250, as described under ``Materials and
Methods.'' The mutated and the parent constructs were used in
parallel transfections of fibroblasts, as described in the legend to Fig. 2. After glycerol shock, the cultures were incubated for 3
h in medium containing 1% fetal calf serum and incubated for another 40
h without or with TGF- (10 ng/ml), in the absence or presence of
TNF-
(25 ng/ml). Processing of samples was carried out as
described in the legend to Fig. 2. The various promoter
constructs used were as follows: panel 1, -342COL1A2
promoter/CAT; panel 2, mutated -342COL1A2 promoter/CAT; panel 3, -265COL1A2 promoter/CAT; panel 4, mutated
-265COL1A2 promoter/CAT. A, autoradiograms of a
representative experiment. B, the results, presented as
relative promoter activity, are the mean ± S.D. of three
independent experiments, each performed using duplicate samples, and
are expressed as percent of acetylation of the
[
C]chloramphenicol
substrate.
Additional 5` deletion to position -161 of the promoter
restored some of TGF- responsiveness (
2.5-fold stimulation) (Fig. 2B). This elevation of promoter activity was lost
when the 5` end of our construct was decreased to position -108
of the promoter. Since the activity of the shortest construct was
extremely low (see Fig. 1and related text under
``Results''), we established stably transfected NIH 3T3
fibroblast cultures with the -108/CAT construct. No significant
effect of TGF-
was detected on the promoter activity using this
experimental approach (not shown). It appears, therefore, that an
essential TGF-
-response element is located between residues
-265 and -241 of the COL1A2 promoter, although additional,
yet somewhat secondary, sequences allowing some TGF-
responsiveness may exist downstream from nucleotide -161.
It should be noted
that the binding activity, as determined by the intensity of the
DNA/protein band, of extracts from TGF--treated cells was not
significantly altered as compared with that of control extracts. This
quantitative similarity may, however, be masking changes in both the
content and the transcriptional activity of the bound complexes.
Specifically, the AP-1 complex is a dimer of gene products of the Fos
and Jun families of oncogenes, which have closely related recognition
sites but different transcriptional activities and DNA binding
affinities (reviewed in Vogt and Bos(1990)). For example, whereas c-Jun
is a potent activator of the c-jun and collagenase promoters,
Jun-B is not and inhibits their trans-activation by c-Jun
(reviewed in Mauviel(1993)). To characterize further the protein
complex binding to the -265/-241 region of human COL1A2,
nuclear extracts from TGF-
-treated fibroblast cultures were
incubated with antibodies against c-Jun or Jun-B, prior to detection of
DNA/protein interactions by gel mobility shift assay. As shown in Fig. 4, the antibody against Jun-B (lane 2), but not
that against c-Jun (lane 1), induced a supershift of the
labeled DNA probe, indicating that Jun-B participates in the formation
of the complex that binds to the -265/-241 region of
COL1A2. This result is in agreement with our previous observations that
TGF-
is a potent activator of jun-B expression in
fibroblasts (Mauviel et al., 1993) and further indicates that
certain components of AP-1 are part of the transcription factor binding
to the COL1A2 promoter.
Figure 4:
Effect of antibodies against c-Jun and
Jun-B on nuclear protein binding to the -265/-241 fragment of COL1A2
promoter. Nuclear extracts from TGF--treated fibroblast cultures
(10 µg) were preincubated on ice for 30 min with antibodies for
either c-Jun (Anti c-Jun) or Jun-B (anti-Jun-B) prior
to the binding reaction with a radiolabeled oligonucleotide
corresponding to the -265/-241 fragment of human COL1A2
promoter. The supershifted band is indicated on the right side of the autoradiogram.
Figure 6:
Effect of overexpression of c-jun on TGF--induced up-regulation of COL1A2 promoter activity.
Confluent fibroblast cultures were transfected with 2 µg/plate of
-342/CAT plasmid construct together with 18 µg/plate of
either an empty expression vector pRSVe or a c-jun expression
vector pRSVc-jun, as indicated. Following glycerol shock, the
cells were placed in DMEM supplemented with 1% fetal calf serum. Three
hours later, TGF-
(10 ng/ml) was added, and the incubation was
continued for another 40 h. CAT activity, representing the activity of
the COL1A2 promoter, was determined, and autoradiograms of a
representative experiment are shown, together with their corresponding
values of CAT acetylation.