(Received for publication, December 11, 1996, and in revised form, June 2, 1997)
From the Brookdale Center for Developmental and
Molecular Biology and ¶ Department of Pediatrics, Mount Sinai
School of Medicine, New York, New York 10029 and the
§ Department of Internal Medicine, National Kanazawa
Hospital, Kanazawa 920, Japan
It is currently debated whether AP1 or Sp1 is the
factor that mediates transforming growth factor 1 (TGF-
)
stimulation of the human
2(I) collagen (COL1A2) gene by
binding to an upstream promoter element (TbRE). The present study was
designed to resolve this controversy by correlating expression of
COL1A2, AP1, and Sp1 in the same cell line and under different
experimental conditions. The results strongly indicate that Sp1 is
required for the immediate early response of COL1A2 to
TGF-
and AP1 is not. The Sp1 inhibitor mithramycin blocked
stimulation of
2(I) collagen mRNA accumulation by TGF-
,
whereas the AP1 inhibitor curcumin had no effect. Furthermore, antibodies against Jun-B and c-Jun failed to identify immunologically related proteins in the TbRE-bound complex, irrespective of whether they were purified from untreated or TGF-
-treated cells. AP1 did
bind to the TbRE probe in vitro, but only in the absence of the upstream Sp1 recognition sequence. Based on this finding and DNA
transfection results, we conclude that the AP1 sequence of the TbRE
represents a cryptic site used under experimental
conditions that either eliminate the more favorable Sp1 binding site or
force the balance toward the less probable. Finally, a combination of cell transfections and DNA-binding assays excluded that COL1A2 transactivation involves the retinoblastoma gene product (pRb), an activator of Sp1, the pRb-related protein p107, an inhibitor of Sp1,
or the Sp1-related repressor, Sp3.
Type I collagen is the major structural component of connective
tissue and consequently one of the key contributors to embryonic development and growth and to tissue repair and homeostasis (1, 2).
Deregulated type I collagen synthesis is invariably associated with
disorders characterized by excessive matrix deposition or abnormal
matrix degradation (3, 4). Hence, transcription of the genes coding for
the 1 and
2 subunits of type I collagen is critical to many
physiological activities and pathological processes. During the past
few years, substantial effort has been devoted toward understanding the
molecular mechanisms that control transcription of the type I collagen
genes (5, 6). Some of the studies have examined tissue specific
expression of the murine
1(I) and
2(I) collagens in
transgenic animals; others have focused on the response of the
human genes to cytokines. Altogether, the analyses suggest that the
regulatory sequences of these coordinately expressed genes are
organized somewhat differently. On the one hand, transcription of the
murine
1(I) collagen gene in distinct mesenchyme lineages appears to
be under the control of separate cis-acting elements
scattered within 3.5 kilobase pairs of upstream sequence (7, 8). On the
other hand, spatiotemporal specificity of the mouse
2(I) collagen
(Col1a2) gene seems to be confined to the
350-bp1 proximal promoter and
cis-acting elements with redundant GC-rich binding sites for
nuclear proteins (9-12).
One of the cis-acting elements of the mouse 350-bp promoter
has been shown to participate in 2(I) collagen expression in dermal
and tendon fibroblasts, and to mediate the transcriptional response of
the gene to transforming growth factor-
1 (TGF-
) (9, 10, 13).
Multiple copies of the
315 to
284 sequence of the Col1a2
gene were sufficient to drive transcription from the basal 40-bp
promoter in transfected fibroblasts and only in the tail and skin of
transgenic mice (9, 10). The finding was in line with prior results,
which had correlated mutations introduced within the
315 to
284
sequence with a 10-fold loss of promoter activity (14). The same region
had also be found to mediate the stimulatory effect of TGF-
through
a CTF/NF-I sequence by an as yet undetermined mechanism (13).
Subsequent work could not confirm the contribution of the CTF/NF-I
binding site to TGF-
responsiveness and instead implicated AP1
without, however, documenting where the factor binds or what the
transactivating mechanism might be (15). A more recent report has made
a correlative argument to link CTF with the antagonistic signals of
TGF-
and tumor necrosis factor-
(TNF-
) on Col1a2
gene expression (16). Work on the human
2(I) collagen
(COL2A1) gene has reiterated the importance of the
corresponding
330 to
283 promoter region in controlling fibroblast
specificity and in mediating TGF-
responsiveness (17-19). It has
also raised the same controversy as the mouse studies did. Whereas
there is general agreement excluding the participation of CTF/NF-I in
the regulation of the human gene, different reports have involved
distinct cis-acting elements and trans-acting
factors in constitutive and TGF-
-stimulated transcription of
COL1A2 (18-20).
One set of studies has shown that the TGF- and TNF-
signaling
pathways converge on the same transcriptional complex bound to the
330 to
255 region of the COL1A2 promoter (18,
21). The sequence consists of two nearly juxtaposed footprints (Boxes A
and B, see Fig. 1), the most distal of which was further subdivided into two (Boxes 5A and 3A) by the following criteria. Deletion of Box
5A increased promoter activity, whereas nucleotide substitutions in Box
3A nearly abrogated it; additionally, distinct nuclear proteins bound
Boxes 5A and 3A independently of each other. Although Box 5A contains
the counterpart of the murine CTF/NF-I binding site (Fig. 1),
recombinant NF-I failed to recognize the human sequence; furthermore,
deletion of Box 5A had virtually no effect on the ability of TGF-
to
stimulate the collagen promoter (18). TGF-
responsiveness was
instead linked to Boxes 3A and B (TbRE) and the cognate nuclear protein
complex (TbRC). Additional in vitro evidence indicated that
one of the TbRC components is the ubiquitous activator Sp1 (18). Most
importantly, it was noted that TGF-
stimulation of COL1A2
gene expression translated into increased intensity of the TbRC in the
electrophoretic mobility shift assay (EMSA) (18). A subsequent study
suggested that tyrosine dephosphorylation may be an obligatory step in
TbRC transactivation (22). These observations led to the formulation of
the following hypothetical mechanism for TGF-
induction of COL1A2
transcription. TGF-
transactivates the TbRC by modifying an Sp1
co-factor through a tyrosine dephosphorylation-dependent
nuclear step. In turn, the post-translational modification increases
either the recruitment of TbRC to the cognate binding site or the
affinity of the prebound complex. It was also hypothesized that the
antagonistic signal of TNF-
affects both TbRC and C1R, the alleged
repressor that binds to Box 5A (21); the conclusion was based on the
observation that overexpression of c-jun blocked cytokine
stimulation of the co-transfected collagen promoter (19).
The above findings have been subsequently challenged by another study
which located the TGF--responsive element to an AP1 recognition
sequence overlapping the 3
end of Box B (Fig. 1) (19). The study has
also suggested that transcriptional stimulation of COL1A2 by TGF-
is
due to a switch in Fos partners, from c-Jun to Jun-B, resulting from
TGF-
induction of jun-B gene expression (19, 23). The
idea has received further support from the independent finding that
overexpression of jun-B induced transcription of the mouse
Col1a2 gene, and antisense jun-B RNA attenuated
its stimulation by TGF-
(15). It should be noted that another study similarly implicated TGF-
induction of jun-B expression
in the down-regulation of the matrix metalloprotease-1 gene in cultured dermal fibroblasts (24). Hence, TGF-
may modulate matrix remodeling by changing the composition of a transcriptional complex that regulates
expression of collagen and collagenase genes in opposite ways (19, 23,
24).
It was the principle objective of the present study to resolve the
above controversy by elucidating whether the factor involved in TGF-
stimulation of COL1A2 transcription is Sp1 or AP1. To this
end, we investigated how different experimental conditions affect
expression of the candidate regulators and the target gene in the same
fibroblast cell line. Within the limitation of this in vitro
experimental model, the results excluded AP1 participation in the early
response of COL1A2 to TGF-
and, indirectly, confirmed Sp1
involvement in this process. They also excluded roles for availability
of active Sp1 and release of Sp3 in TbRC transactivation.
TGF-1 and Yersinia-derived
protein-tyrosine phosphatase were purchased from Boehringer Mannheim.
Curcumin, mithramycin A, cycloheximide and phorbol 12-myristate
13-acetate (PMA) were obtained from Sigma. Oligonucleotides containing
consensus recognition sequences for Sp1 and GATA factors and for wild
type and mutant AP1, as well as antibodies against Sp1 (PEP-2), c-Jun
(D and N), Jun-B (N-17), pRb (C-15), and p107 (SD9) were purchased from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Additional antibodies against Sp1 and Sp3 were generously provided by Dr. J. Horowitz (Duke
University Medical Center, Durham, NC). The Rb expression vector, pHRb,
and the parental plasmid, pBJ1874, were a kind gift of Dr. R. Weinberg
(Whitehead Institute for Biomedical Research, Cambridge, MA); details
about the construction of the COL1A2/CAT chimeric plasmids have been
published (17, 18). cDNA probes for c-jun and
jun-B were kindly provided by Dr. T. Curran (Roche Institute
of Molecular Biology, Nutley, NJ), whereas the Sp1 cDNA was a
generous gift of Dr. J. T. Kadonaga (University of California, San
Diego, La Jolla, CA). The cDNA for human glyceraldehyde-3-phosphate dehydrogenase was purchased from American Type Culture Collection (Rockville, MD), and the COL1A2 cDNA has been described previously (26).
Primary human fetal
skin fibroblasts (CF-37) were grown in Dulbecco's modified Eagle's
medium containing high glucose (4.5 g/liter) supplemented with 10%
fetal bovine serum (HyClone Laboratories, Logan, UT) (17). Preparation
and transfection of plasmid DNA into cells were performed as described
previously (17). Five hours after transfection, fibroblasts were
treated with 10% glycerol for 90 s, and then placed in medium
containing 0.1% fetal bovine serum. Two hours later, TGF- or PMA
were added to the culture media at the final concentration of 5 ng/ml
or 60 ng/ml. Cells were harvested 48 h later and subjected to
luciferase and CAT assays as described previously (18). In some
experiments, cells were co-transfected with the
378COL1A2/CAT
construct and pHRb or pBJ1874, expression vectors with or without the
Rb gene. In this case, cells were harvested 48 h after
transfection to prepare nuclear extracts and total RNA, and to
determine CAT activities. The Mann-Whitney U test was used
to determine the statistical value of the functional data. The
transcriptional activity of chimeric constructs in transfection assays
was always normalized against the co-transfected pSV-luciferase vector
(Promega Corp., Madison, WI).
Confluent CF-37 fibroblasts were placed in low serum
18 h before TGF- addition. Likewise, cells were preincubated
with curcumin (20 µM) for 30 min or with mithramycin (100 nM) for 18 h prior to TGF-
administration. In some
experiments, cycloheximide and PMA were added at final concentrations
of 30 µg/ml and 60 ng/ml, respectively. Viability of the cells was
estimated using the trypan blue (0.4% in PBS) exclusion test; in all
cases, cellular viability was more than 90%. Total RNA was extracted
4 h after the addition of TGF-
and processed for Northern blot
hybridizations according to standard protocols (25, 27). Likewise, a
standard protocol was used to determine rates of COL1A2
transcription in control and mithramycin-treated cells using
glyceraldehyde-3-phosphate dehydrogenase and pBR322 DNA as controls
(28). Quantitative data were obtained using the computer program
NIH-image software after scanning the autoradiographs with Adobe
Photoshop (Adobe Systems Inc., Mountain View, CA).
Nuclear extracts were
prepared from control and TGF--treated fibroblasts, alone or in
combination with curcumin or cycloheximide. Confluent cells were
maintained for 18 h in low serum prior to the addition of either
TGF-
or PMA. Cells were preincubated with curcumin as described
above. Cells were harvested 3 h after treatment and processed to
prepare nuclear extracts according to the published protocol (29).
Nuclear extracts were used in the EMSA
with COL1A2 oligonucleotides spanning from 313 to
183
(TbRE) and from
271 to
235 (Box B). Although longer than the TbRE
and Box B, the EMSA probes are nevertheless identified by the names of
these cis-acting elements. Relevant to the present study,
the TbRE includes the Sp1 and AP1 recognition sequences and Box B
encompasses only the AP1 binding site (Fig. 1). Oligonucleotides were
end-labeled with [
-32P]dCTP using the Klenow fragment
of DNA polymerase or with [
-32P]dATP and T4
polynucleotide kinase (27). EMSAs were performed as described
previously (18, 29). For mithramycin treatment, DNA probes were
preincubated for 1 h at 4 °C in the presence of the drug (100 nM final concentration) before being added to nuclear extracts. In the competition experiments, unlabeled oligonucleotides were always added at 100-fold molar excess. The TbRC pattern shown in
the present and past studies (18, 21, 22) was reproducibly obtained
only with hyperconfluent cells, and with nuclear proteins purified
according to the protocols of Truter et al. (29) or Andrews
and Faller (30). In the antibody interference assays, 2 µg of
antibodies were preincubated with 5 µg of nuclear extracts for 1 h at 4 °C before adding labeled probes. For the immunodepletion experiments, 20 µg of antibodies were conjugated to protein G-agarose beads in PBS and, after washing, they were incubated for 3 h at 4 °C with 5 µg of nuclear proteins. Following centrifugations, supernatants were incubated with labeled oligonucleotide probes and
analyzed by the EMSA.
Nuclear extracts (15 µg for Sp1 detection and 20 µg for Rb detection) were electrophoresed in a sodium dodecyl sulfate-polyacrylamide gel (-7.5% polyacrylamide) and transferred onto nitrocellulose filters as described by Towbin et al. (31). Filters were probed with antibodies (1:4000 dilution), followed by incubation for 1 h with horseradish peroxidase-conjugated rabbit anti-goat IgG diluted 1:3000. Sp1 and pRb were detected using an enhanced chemiluminiscent system (ECL, Amersham Corporation) according to the manufacturer's recommendations.
Inagaki et al. (18) originally proposed that TGF-
stimulates COL1A2 transcription by modifying the
transactivating potential of an Sp1-containing complex (TbRC) that
binds in vitro to a 130-bp probe (-313/-183) inclusive of
the TbRE (Fig. 1). In contrast, Chung
et al. (19) have recently argued that TGF-
stimulation is
mediated by the cytokine-induced change in the composition of the AP1
complex bound to the
265/
241 sequence from Fos/c-Jun to Fos/Jun-B
(Fig. 1). We have revisited this important issue using a strategy that
took into account the biological implications of both models.
Specifically, we have compared the effects of various experimental
conditions on the expression of the endogenous COL1A2 gene
and the candidate regulators Sp1 and AP1 on the in vitro
binding of the purified nuclear proteins.
It is well established that TGF- stimulates
collagen production by acting in part at the transcriptional level
(32-34). It is also widely appreciated that a variety of factors,
including culture conditions and the target cell, play a role in
determining the precise mode of action of the cytokine and thus, the
nature and extent of the response (35, 36). In view of these
considerations, we first established how TGF-
modulates expression
of the genes coding for
2(I) collagen, Sp1, and Jun-B in our
experimental system, primary fibroblasts (CF-37) derived from human
fetal skin (17).
The AP1 model predicts synthesis of the Jun-B protein as an
intermediate step in TGF- stimulation of COL1A2 (19).
This prediction is consistent with the reported induction of
jun-B mRNA accumulation within 1 h of TGF-
administration in several cell culture systems, including lung
epithelial cells, foreskin fibroblasts and epidermal keratinocytes (34,
35). A rapid and substantial increase of jun-B mRNA
after TGF-
treatment was also seen in our dermal fibroblasts
cultures; like other cell culture systems, the increase was unaffected
by co-administration of cycloheximide at a concentration known to
nearly abolish protein synthesis in fibroblasts (37) (Fig.
2A). A more delayed
stimulation was seen in duplicate Northern blots hybridized to
COL1A2 or c-jun; also in this case, the increases
were not blocked by addition of cycloheximide (Fig. 2A).
TGF-
stimulation of COL1A2 transcription thus appears to occur
independently of de novo synthesis of Jun-B, the step
allegedly responsible for the change in AP1 composition and
consequently, for COL1A2 transactivation (19). The mRNA data also excluded causal association between de novo Sp1
synthesis and COL1A2 stimulation by TGF-
, a finding in line with the
hypothesized co-factor modification of the Sp1 model (Fig.
2A) (18, 22). In addition, they documented 2- and 1.5-fold
increase in Sp1 mRNA and protein content after 6 h of TGF-
treatment, respectively (Fig. 2, A and B).
Mithramycin Blocks TGF-
Curcumin and
mithramycin are inhibitors of AP1 activation and Sp1 binding,
respectively (36, 38). These agents were therefore used to compare the
consequences of blocking the activity of each nuclear factor on
COL1A2 gene regulation. Two different batches of nuclear
extracts were analyzed by EMSA using high affinity recognition
sequences for AP1 and Sp1. The first batch of extracts was prepared
from CF-37 cells cultured with and without TGF- and with and without
curcumin. An aliquot from each fibroblast culture was processed in
parallel for Northern analysis. Since mithramycin inhibits DNA binding
by modifying GC-rich sites, the AP1 and Sp1 oligonucleotides were
incubated in vitro with the drug prior to incubation with
nuclear proteins purified from untreated and TGF-
-treated cells. RNA
samples for Northern analysis were extracted from CF-37 fibroblasts
cultured with mithramycin under conditions comparable to the in
vitro treatment.
EMSAs confirmed the specificity of the two drugs in the selective
elimination of either Sp1 or AP1 binding (Fig.
3A). Northern hybridizations
established a causal association between inhibition of Sp1 binding and
loss of COL1A2 stimulation by TGF- (Fig. 3B). More
importantly, they documented that curcumin inhibition of AP1 activity
affects neither COL1A2 basal expression nor its stimulation by TGF-
(Fig. 3B). Consistent with the reported role of Sp1 in maintaining constitutive COL1A2 expression (39),
2(I) collagen mRNA levels decreased about 30% in uninduced cells after
mithramycin treatment (Fig. 3B). Nuclear run-on experiments
confirmed that mithramycin acts mostly by repressing COL1A2
transcription (Fig. 3C). The results therefore strongly
implicated Sp1 in the in vivo modulation of
COL1A2 gene expression by TGF-
, while concomitantly excluding AP1 from this process.
Sp1 Binding to the TbRE Is Favored over AP1
The hypothesis of
a TGF--induced switch in the composition of the AP1 complex is
chiefly based on the observation that Jun-B and not c-Jun antisera
affect in vitro binding to the
265/
241 sequence of
nuclear proteins from stimulated cells (19). Here, the same test was
repeated using CF-37 fibroblasts and probes that include either the AP1
binding site of Box B (probe
265/
241) or the AP1 and Sp1
recognition sequences of the TbRE (probe
313/
183). The former is
the same probe used in the study that has led to the formulation of the
AP1 model (19). In agreement with the mRNA expression levels (Fig.
2), the amount of Jun-B bound to the
265/
241 probe increased
following TGF-
treatment (Fig. 4A). The c-Jun antisera
interfered with AP1 binding almost as effectively in control as in
TGF-
-treated samples (Fig. 4A). This last result is in
complete disagreement with the finding of Chung et al. (19),
who reported the absence of immunologically identifiable c-Jun in the
AP1 complex of TGF-
-induced cells. Unfortunately, this discrepancy
can not be resolved since the authors of that report did not examine
the composition of AP1 in uninduced cells (13). The results of the
immunointerference assays using the TbRE probe further questioned the
validity of the AP1 model.
Despite including the 265/
241 sequence and thus the AP1 site, the
TbRE probe bound a complex without immunologically identifiable Jun
subunits irrespective of whether the extracts were derived from
untreated or TGF-
-treated cells (Fig. 4B). Consistent
with previous findings (18), the Sp1 antisera eliminated binding of the
top two TbRC bands (compare the first four lanes with the last four lanes of Fig. 4B). The results
therefore indicated that AP1 can not bind to the cognate site in the
presence of Sp1. We also noted that cycloheximide did not block the
TGF-
induced TbRC binding (Fig. 4B). This observation
further strengthened the positive correlation we had already
established between the responses to TGF-
of the TbRC and the
endogenous COL1A2 gene. A positive correlation was also
established in mithramycin-treated cells between inhibition of
endogenous COL1A2 gene expression and loss of binding to the
313/
183 probe (Figs. 3B and 4C). The
correlation was substantiated by the finding that curcumin affected
neither accumulation of
2(I) collagen mRNA nor binding to the
313/
183 probe (Figs. 3B and 4C). We will
discuss later the possible reasons behind the elimination of the whole
TbRC by the Sp1 antisera in the mithramycin-treated sample.
Since AP1 could bind the site overlapping Box B under particular
in vitro conditions, we thought the same may also happen in
transient transfection experiments. To this end, we compared the
response of the transfected 378COL1A2/CAT plasmid and of the
endogenous COL1A2 gene in CF-37 cultures treated with PMA, an AP1 inducer and a collagen inhibitor. The comparison documented the
paradoxical result of PMA up-regulation of the former and down-regulation of the latter (Fig. 5).
As predicted by the above experiments, curcumin blocked PMA-induced
down-regulation of the endogenous COL1A2 gene (Fig.
5B). On the other hand, mutation of the AP1 site overlapping
Box B provided a direct linkage between this sequence and the
unphysiological response of the transfected plasmid to PMA (Fig.
5A). In agreement with the noted contribution of Box B to
basal COL1A2 transcription (18), the mutant plasmid was also
less active than the wild type counterpart (Fig. 5A). Collectively, these experiments suggested that AP1 may bind the cognate
site of the TbRE but only under experimental conditions that adversely
influence binding of Sp1.
Sp1 Is Not the Limiting Factor in COL1A2 Transactivation
The
evidence described so far was overwhelmingly in support of Sp1
involvement for the early response of COL1A2 to TGF-. Although a
hypothetical mechanism has been proposed to explain how the
Sp1-containing complex induces COL1A2 transcription, the details of it are far from being clear. For example, TGF-
could facilitate TbRC formation indirectly, by releasing an inhibitor from
one of its components or directly, by changing its composition. We
explored these possibilities as they apply to Sp1, the only known
component of the TbRC.
It has been shown that TGF- dephosphorylates the retinoblastoma
protein, pRb (40); it has also been reported that pRb stimulates Sp1
transactivation either by liberating it from a specific inhibitor or by
interacting directly with Sp1 (41-43). It was therefore formally possible that in our system TGF-
may increase the pool of active Sp1 molecules via pRb dephosphorylation. We investigated
this possibility by examining the consequences of Rb overexpression on
the endogenous COL1A2 gene and the transfected
378
promoter. To this end, CF-37 fibroblasts were co-transfected with the
378COL1A2/CAT construct and the pHRb or the pBJ1874 plasmid (22).
Transfected cultures were processed in parallel for protein and RNA
analyses and CAT assays. Western analysis documented the production of larger amounts of pRb in the pHRb-transfected compared with the pBJ1874-transfected cells and showed that most of it was
dephosphorylated (Fig. 6A).
Unlike the slight increase of c-jun, Northern analysis revealed that
2(I) collagen mRNA levels remained virtually
unchanged in Rb-overexpressing fibroblasts compared with control cells
(Fig. 6B). Likewise, the CAT assays illustrated that Rb
overexpression had no significant impact on the relative stimulation of
the
378 promoter by TGF-
(Fig. 6C). Therefore, Rb seems
not to play a role in TGF-
stimulation of COL1A2
transcription.
A candidate for Sp1 inhibition is the pRb-related protein p107 (44). To
assess the potential involvement of p107, as well as to independently
confirm the above conclusion, nuclear extracts from untreated cells
were depleted of p107 or Rb using specific antibodies coupled to
agarose beads. As a positive control, nuclear extracts were
immunodepleted with Sp1 antisera. The unbound material was then
incubated with the TbRE, with or without prior treatment with a
tyrosine phosphatase enzyme. Finally, the resulting EMSA patterns were
compared with those of nuclear extracts which had not been subjected to
immunodepletion (Fig. 7A). In
designing these experiments, we assumed that the change of EMSA pattern observed with nuclear extracts from TGF--treated cells was
functionally equivalent to that seen with nuclear extracts incubated
in vitro with tyrosine phosphatase (22).
The experiments documented that the relative intensity of the TbRC
remained virtually the same in controls and in samples immunodepleted
of pRb or p107 (Fig. 7A). In contrast, immunodepletion of
Sp1 resulted in the disappearance of the whole TbRC (Fig.
7A). To exclude that loss of TbRC binding might be due to
proteolysis, the same Sp1-immunodepleted sample was incubated with Box
B. This documented retention of AP1 binding activity and thus inferred integrity of the nuclear extract (Fig. 7B). In conclusion,
in vitro treatment of nuclear extracts with protein-tyrosine
phosphatase increased the intensity of the TbRC irrespective of p107
and pRb but not of Sp1. A last immunointerference experiment examined the potential involvement of the Sp1-related inhibitor, Sp3, in TGF-
stimulation of COL1A2 (45). Comparison of the EMSA patterns obtained
after preincubation with specific antibodies against Sp3 revealed some
difference in relative content of Sp3 between unstimulated and
stimulated nuclear extracts (Fig. 8).
However, the difference was not as dramatic with the Sp1 antisera and
certainly not enough to support the idea of such a switch in TbRC
composition as the major mechanism for COL1A2 stimulation. Altogether,
the results of these analyses excluded that availability of active Sp1
and release of Sp3 from the TbRC may play major roles in TGF-
stimulation of COL1A2 gene expression.
The pathways leading to TGF- regulation of genes involved in
matrix assembly and remodeling, as well as the transcriptional mechanisms underlying them, are largely unknown. A case in point is the
cytokine-induced stimulation of the major structural component of
connective tissue, type I collagen. In the past few years, different
lines of investigation have yielded conflicting results regarding
TGF-
up-regulation of COL1A2 transcription. The
controversy centers around the identity of the cis-acting
element and trans-acting factor mediating most of the
cytokine-induced response from the proximal promoter sequence. As a
result, two distinct models for TGF-
modulation of COL1A2 activity
have been proposed (18, 19).
In its simplest formulation, the first proposal envisions the cytokine
triggering an intracellular signal, which ultimately leads to tyrosine
dephosphorylation of a nuclear protein(s) that in turn modifies the
transactivating potential of an Sp1-containing complex (TbRC). The
model was based on the following lines of evidence (18, 22). First,
mutations in the TbRE region reduce TGF- responsiveness of the
transfected
378 COL1A2/CAT plasmid. Second, the entire TbRE but
neither Box 3A nor Box B alone, confers TGF-
responsiveness to an
otherwise unresponsive heterologous promoter. Third, Sp1 competitors or
Sp1 antisera eliminate binding of part of the TbRC to the GC-rich
sequence of Box 3A. Fourth, in vitro binding of nuclear
proteins to the TbRE but neither Box 3A nor Box B alone, increases
after TGF-
administration to cell cultures. Fifth, in
vivo inhibition of tyrosine phosphatase activity correlates with
block of COL1A2 stimulation by TGF-
and with loss of TbRC binding;
conversely, inhibition of tyrosine kinase activity has opposite
effects.
The alternative proposal hypothesizes that TGF- stimulates
jun-B expression and this in turn induced COL1A2
transcription by changing the composition of AP1 from Fos/c-Jun to
Fos/Jun-B (19). The lines of supportive evidence are the following.
First, mutations of the TbRE region suggest that the major responsive sequence resides within a smaller segment of the TbRE comprising nucleotides
265 to
241. Second, AP1 recognizes the
265/
241 sequence in in vitro binding assays. Third, the AP1 complex
purified from TGF-
-stimulated cells apparently contains Jun-B but
not c-Jun. Fourth, overexpression of c-jun in neonatal
foreskin cultures inhibits TGF-
stimulation of the
342 promoter.
The AP1 model is mostly based on correlative findings rather than on
directly testing how jun-B overexpression affects COL1A2
expression or what the c-Jun content is in the unstimulated AP1
complex. The former point was addressed in a parallel study of the
mouse Col1a2 gene, which indirectly corroborated the AP1
model of TGF-
stimulation (15). The study in fact showed that
overexpression of jun-B in NIH-3T3 fibroblasts increases the
activity of the co-transfected 350-bp Col1a2 promoter nearly
100-fold. However, the same report also showed that c-jun
overexpression stimulated COL1A2 transcription about
4-fold.
We believe that results presented here build a strong and compelling
case in favor of the Sp1 over the AP1 model. Addition of mithramycin or
curcumin to fibroblast cultures revealed that the former (and not the
latter) inhibits constitutive and TGF--induced transcription of
COL1A2. Furthermore, cycloheximide had no effect on TGF-
stimulation of either
2(I) collagen or jun-B
mRNA accumulation, thus excluding that induction of the former gene
depends on synthesis of the latter product. Together, these results
rule out AP1 involvement and bring into question the relevance of the
c-Jun to JunB switch. They also point to the strict requirement of
GC-rich regulatory elements for proper COL1A2 expression. The
conclusion is in line with recent evidence showing that mithramycin
addition to human primary fibroblasts reduce constitutive and
TGF-
-induced transcription of the coordinately expressed
1(I)
collagen gene (46). Moreover, COL1A2 dependence on GC-rich
regulatory elements is consistent with recent work that has documented
their critical contribution to tissue specificity (9, 12, 20, 28, 46).
The causal relationship between the GC-rich binding site of the TbRE
and the mithramycin block of COL1A2 stimulation is solidly founded on
the independent mapping of the TGF-
-responsive element within this
segment of both the human and mouse promoters (13, 18, 19).
The TbRE binds in vitro Sp1 and not AP1. This was
demonstrated by a variety of tests, which included immunointerference
and immunodepletion assays using specific Sp1 and AP1 antibodies, as
well as treatment with Sp1 and AP1 inhibitors. Collectively, they
suggest that AP1 and Sp1 binding to the TbRE are mutually exclusive,
and that the latter is normally favored over the former. We have
corroborated this claim by documenting the paradoxical responses to PMA
of the endogenous gene and of the transfected promoter. Implicitly, the
result provides an alternative interpretation of the co-transfection
experiments upon which the AP1 model has been built (18). Accordingly,
we suggest that the cryptic AP1 sequence overlapping Box B
is used under artifactual conditions which either eliminate the more
favorable binding site (Box B without Box 3A) or force the balance
toward the less probable (AP1 overexpression). We cannot, however,
exclude the possibility that AP1, alone or in cooperation with the
TbRC, may participate in COL1A2 regulation at some later time during
TGF- stimulation, under different physiological or tissue culture
conditions, or in different cells.
The above results are also worthy of a few additional comments. The
first pertains to the different effect that Sp1 immunointerference has
on TbRC binding compared with the Sp1 immunodepletion and the
mithramycin treatment (compare relevant samples of Figs. 4B and 8 with those of Figs. 4C and 7). The difference, loss of
Sp1 versus loss of both Sp1 and Cx, may simply reflect
experimental idiosyncrasies. Alternatively, it may indicate the strict
requirement of Sp1 for TbRC formation and/or stabilization. In other
words, one could reasonably argue that the last two treatments are more effective than immunointerference in blocking Sp1 availability (immunodepletion) or binding (mithramycin treatment) and, consequently, in eliminating the whole TbRC. The proposal is supported by previous competition experiments, which suggested the existence of stabilizing interactions among the TbRC components, particularly between Sp1 and
Cx, the ill defined factor(s) that interacts with Box B (18). Unfortunately, activation of the cryptic AP1 site
overlapping Box B has hampered characterization of Cx and thus,
clarification of this important point. The second comment regards the
discrepancy between the cell transfection data responsible for the
formulation of the two opposing models for TGF- stimulation of
COL1A2 transcription (18, 19). Our experiments documented
TGF-
responsiveness of a chimeric construct driven by a promoter
fragment that includes the AP1 binding site of Box B, but lacks the
upstream Sp1 recognition sequence of Box 3A (18). However, the
induction was statistically less significant than the one obtained with
constructs harboring either Boxes A and B, or Boxes 3A and B (18).
Chung et al. (19) reported maximal TGF-
responsiveness
with a 5
deletion construct retaining only the most proximal of the
three Sp1 binding sites of the TbRE. We have no explanation for these
differences, short of noting that other investigators have also
experienced greater loss than Chung et al. (19) in the
constitutive activity of the COL1A2 promoter (10-fold
versus 2-fold) when mutations were introduced in the Sp1
recognition sequence of Box 3A (20). The third and final comment is in
reference to the results of the PMA experiments. Curcumin inhibition of
COL1A2 down-regulation by PMA indicates that the signal is ultimately
transduced through AP1. On the other hand, the responses of the
transfected promoter and endogenous gene strongly suggest that the
PMA-responsive element of COL1A2 resides outside of the
378 sequence. Together the results infer that AP1 access to the Box B
site of the endogenous gene in PMA-treated cells is conceivably
hindered by chromatin and/or interactions between the TbRE and other
critical regulatory elements, such as the recently described
far-upstream enhancer of Col1a2 (47).
The present study has also extended the analysis of the potential
factors and mechanisms responsible for TbRC transactivation. Previous
work has suggested that tyrosine dephosphorylation of nuclear proteins
is an intermediate step in TGF- stimulation of COL1A2 expression
(22). It is, however, unknown if protein dephosphorylation is triggered
by increased phosphatase activity or decreased kinase activity. It is
also yet to be determined whether the visual change detected by EMSA
reflects binding of more TbRC molecules or increased affinity of the
prebound complex. Finally, it is yet to be determined what the
mechanism in each of these situations might be. We have explored these
possibilities as they apply to Sp1, the only known component of the
TbRC. Far from being exhaustive, the analysis has nevertheless
eliminated some obvious candidates and strengthened our belief that a
novel Sp1 partnership is responsible for TbRC transactivation by
TGF-
.
In conclusion, we have attempted to resolve the controversy surrounding
the identity of trans-acting factors and
cis-acting elements and, implicitly, the nature of the
molecular mechanisms responsible of the transcriptional response of
COL1A2 to TGF- in cultured fibroblasts. Within the
limitations of the in vitro model, we believe that we have
presented convincing evidence in favor of our early model. This
postulates that modification of an Sp1-containing complex is the last
step in the pathway that transduces the TGF-
signal from the cell
surface to the COL1A2 gene. We also believe we have
convincingly excluded the alternative model, which suggests that the
TGF-
signal is elaborated transcriptionally through a change in AP1
composition. In so doing, we have offered an experimental explanation
for the discrepancy between the studies supporting each of the two
models. We are nevertheless aware of the intrinsic limitations of our
method of analysis and of our experimental system, and understand that
ultimate proof of the model can only be reached after full biochemical
characterization of the TbRC. Work in progress is aimed at achieving
this important goal.
We thank Dr. S. Tanaka for many helpful discussions; Drs. T. Curran, J. Horowitz, J. T. Kadonaga, and R. Weinberg a for providing critical reagents; and Karen Frith for typing the manuscript.