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INTRODUCTION |
As TGF-
1 promotes
fibroblast proliferation and matrix synthesis, this cytokine has long
been proposed to initiate and maintain fibrosis (e.g. Refs.
1-3). Indeed in acute drug- or surgery-induced animal models of
fibrosis, neutralizing TGF-
action reduces the deposition of collagen (for a review, see Ref. 1). Much interest has
been devoted to developing anti-TGF-
strategies, such as neutralizing anti-TGF-
antibodies or soluble TGF-
receptor
fragments, to combat fibrosis; however, as TGF-
possesses multiple
roles in human physiology (4, 5), generally blocking its effects might
be expected to have deleterious effects clinically. Thus, identifying
downstream mediators of the profibrotic effects of TGF-
might
be useful in developing more clinically appropriate antifibrotic strategies.
Accordingly much interest has been recently devoted to the TGF-
target gene CTGF. CTGF, a secreted protein (6) initially identified in
the conditioned medium of cultured endothelial cells (7), is a member
of the CCN (CTGF, Cyr61, and
Nov) family of proteins that promote angiogenesis, cell
migration, and cell adhesion (8). Recently CTGF knockout mice have been
generated; mice homozygous for a deletion of the CTGF gene die soon
after birth due to a defect in skeletal development characterized, in
part, by reduced expression of bone-specific matrix genes (9).
Pathologically CTGF seems to contribute to fibrotic disorders by
mediating at least some of the profibrotic effects of TGF-
(10). A
CTGF response element exists in the type I collagen promoter (11). Furthermore, in mice, subcutaneous injection of TGF-
results only in
a transient fibrotic response; however, co-injection of CTGF and
TGF-
results in sustained, persistent fibrosis (12). Thus, CTGF and
TGF-
seem to synergize to promote chronic fibrosis. This phenomenon
may result from the inherent stickiness of CTGF, which may enhance the
activity or stability of TGF-
at low TGF-
concentrations (13).
Recently we found that a small molecule inhibitor, Iloprost, reduces
CTGF expression in vitro and in vivo and
alleviates symptoms of fibrosis in vivo (14, 15). Thus understanding how to control CTGF expression would seem to be important
in developing novel antifibrotic therapies (16).
In skin, CTGF is not normally expressed; however, in dermal fibroblasts
exposed to TGF-
, CTGF mRNA and protein expression are induced
(17-21). The induction of CTGF by TGF-
is cell-type specific, for
example, occurring in connective tissue cells but not in epithelial
cells (17-22). The regulation of CTGF expression by TGF-
appears to
be controlled primarily at the level of gene transcription (16-22).
The induction of CTGF mRNA by TGF-
is rapid, occurring within 30 min of ligand addition, and does not require de novo protein
synthesis as this induction occurs even in the presence of an inhibitor
of protein synthesis (17).
TGF-
induction of gene expression has been extensively studied and
generally involves the action of members of the Smad family of proteins
(23). Activation of TGF-
-mediated gene expression is generally
mediated through Smad 2, 3, and 4. Smad 2 and 3 are normally present in
the cytosol. Once activated by TGF-
, Smad 2 and 3 become
phosphorylated by type I TGF-
receptor kinase and then, after
forming a complex with Smad 4, translocate to the nucleus and activate
expression of target genes. The inhibitory Smads, Smad 6 and 7, negatively regulate this process. Recent studies have identified
a Smad-responsive promoter element, GTCTAGAC, that binds Smad 3 and 4 (23). Recently we identified a functional consensus Smad binding site
in the CTGF promoter that is essential for the TGF-
induction of
CTGF in fibroblasts (20, 21). Cotransfection of Smad 3 and 4 enhanced
CTGF expression significantly (20). TGF-
induction of CTGF did not
occur in embryonic fibroblasts cultured from Smad 3 knockout mice (20),
suggesting that TGF-
induction of CTGF occurs in a Smad
2-independent fashion. Intriguingly the Smad binding site of CTGF is
insufficient to confer TGF-
responsiveness to a heterologous
promoter (21), suggesting that additional as yet unidentified factors
must be required for the TGF-
-mediated induction of CTGF gene
expression in fibroblasts.
In this report, we elucidate requirements necessary for the TGF-
induction of CTGF in fibroblasts but not in epithelial cells. We
identify cis-acting promoter sequences and signaling pathways involved
with this induction. To our knowledge this is the first investigation
of the regulation of a fibroblast-selective TGF-
-responsive promoter. Our results give new insights into fibroblast-selective TGF-
signaling and suggest methods of developing new antifibrotic therapeutic strategies.
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MATERIALS AND METHODS |
Cell Culture, Transfections, and DNA Constructs--
Human
dermal and mouse NIH 3T3 fibroblasts were cultured as described
previously (19, 20). MvLu epithelial cells were purchased (ATCC)
and cultured in 10% Dulbecco's modified Eagle's medium, 10% fetal
bovine serum (Cellgro). Cells were transfected with LipofectAMINE plus
(Invitrogen) or FuGENE (Roche Molecular Biochemicals) as described by
their manufacturers. Compounds used (Go 6850, Go 6976, Go 6983, U0126,
SB203580, and SP600125 (all from Calbiochem)) were added at the
concentrations indicated 45 min before adding TGF-
2 (Celtrix) for
24 h. Expression vectors (1 or 0.5 µg as indicated) encoding
dominant negative Ras (N17, Upstate Biotechnology), active MEKK1
(Stratagene), c-Jun (R. Tjian, University of California-Berkeley),
dominant negative c-Jun (TAM67, M. J. Birrer, NCI, National
Institutes of Health, Bethesda, MD), dominant negative MKK4 (R. Davis,
University of Massachusetts Medical School, Worcester, MA), YAP65
(Research Genetics, GenBankTM accession number AW215560),
or empty expression vector were transfected into each well of a
six-well plate along with a full-length CTGF promoter/SEAP
reporter expression vector (containing a promoter fragment between
805 and +17 of the CTGF promoter, 0.5 µg; Ref. 14) and
CMV-
-galactosidase (0.25 µg, Clontech) as an
internal transfection control. An additional reporter construct
containing multiple copies of the consensus Smad recognition sequence
subcloned upstream of the luciferase reporter gene (SBE-lux) was
from P. Ten Dijke (Ludwig Institute, Uppsala, Sweden). Promoter assays were performed and standardized to
-galactosidase as described (Tropix). Values shown are the average (±S.D.) of at least three replicates and at least two independent trials. Statistical analysis was performed using the Student's t test (p < 0.05). A DNA construct containing nucleotides
805 to
23 of the
CTGF promoter subcloned upstream of the herpes simplex
virus minimal thymidine kinase (TK) promoter and SEAP reporter gene was
as described previously (21). Additional promoter deletion
constructs were generated using Pfu polymerase (New England
Biolabs) and appropriate synthetic oligonucleotide primers (Sigma
Genosys). The resultant DNA fragments were subcloned into TK-SEAP
(Clontech). Linker scanning and TEF binding motif
mutations were performed using oligonucleotides containing appropriate
mutations (Sigma Genosys) and a mutagenesis kit (Stratagene). All
constructs were fully sequenced before use. For Western blot analysis,
a rabbit polyclonal anti-CTGF antibody was used as described previously
(19), and a mouse anti-
-tubulin antibody (Sigma) was utilized.
Gel Shift Analysis--
Nuclear extracts were prepared using a
kit (Pierce), and protein concentration was determined (Bio-Rad). A
double-stranded annealed oligomer spanning nucleotides
126 to
77 of
the CTGF promoter (Sigma Genosys) was labeled with
[32P]ATP (PerkinElmer Life Sciences) and polynucleotide
kinase (New England Biolabs) and used as probe (60,000 cpm/reaction).
As competitor oligomers, a 100-fold excess of wild-type probe or
oligomers otherwise identical to the wild-type probe but bearing
mutations in either TEF recognition element were used.
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RESULTS |
TGF-
Induces CTGF Expression in Fibroblasts but Not in
Epithelial Cells--
To verify previously published data showing
that the TGF-
induction of CTGF protein occurred in
fibroblasts but not in epithelial cells (17), we cultured human
foreskin fibroblasts or MvLu epithelial cells until confluence. Cells
were then serum-starved for 18 h and treated with or without 25 ng/ml TGF-
2 for an additional 24 h. Cells were then harvested,
and the resultant protein extracts were subjected to Western blot
analysis with a rabbit anti-CTGF antibody (Fig.
1A, cell layer).
Equal amounts of media were also subjected to Western analysis (Fig.
1A, media). As anticipated, in the absence of
exogenously added TGF-
, neither cell type expressed CTGF. However,
TGF-
2 induced CTGF protein expression in fibroblasts but not in MvLu
epithelial cells.

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Fig. 1.
TGF- 2 induces CTGF
protein (A) and CTGF promoter (B)
activity in fibroblasts but not in epithelial cells.
A, human foreskin fibroblasts (HFF) and
MvLu epithelial cells were serum-starved for 18 h prior to
incubation with or without 25 ng/ml TGF- 2 for an additional 24 h. Cell layers were harvested, and the resultant protein extracts (25 µg/lane) were subjected to Western blot analysis with a rabbit
anti-CTGF antibody (19) or an anti- -tubulin antibody to verify equal
protein loading among samples. Equal amounts of conditioned media were
also subjected to Western blot analysis with an anti-CTGF antibody.
B, a construct containing a full-length CTGF promoter
(spanning nucleotides 805 to +17) subcloned in front of the SEAP
reporter gene or a construct containing 12 copies of a Smad binding
element (CAGA) upstream of the luciferase reporter gene (1 µg/well of
a six-well plate) was transfected into the cells described in
A. After an 18-h serum starvation step, cells were treated
with or without 25 ng/ml TGF- 2 for an additional 24 h. Cells
were cotransfected with a control CMV promoter-driven -galactosidase
expression plasmid (0.25 µg/well), which was used to adjust for
differences in transfection efficiencies among samples. Average
expression values (±S.D., n = 6) are shown. Results
similar to those obtained with human foreskin fibroblasts were observed
in NIH 3T3 fibroblasts (not shown).
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To assess whether TGF-
induction of CTGF was conferred by elements
in the CTGF promoter, we transfected into cells a DNA construct
containing a CTGF promoter fragment spanning nucleotides
805 and +17
that was subcloned in front of the SEAP reporter gene (19, 20). After
an 18-h serum starvation step, cells were exposed for 24 h to 25 ng/ml TGF-
2. Media were then assayed for SEAP expression. We found
that TGF-
potently induced CTGF promoter activity in fibroblasts but
not in MvLu cells (Fig. 1B). These results were consistent
with previously published observations using a slightly larger CTGF
promoter fragment (17). MvLu cells were capable of inducing a
Smad-dependent transcriptional response to TGF-
as
TGF-
was able to induce expression of a transiently transfected
construct expressing a luciferase reporter gene under the control of
multimers of a consensus Smad recognition sequence (SBE-luciferase;
Fig. 1B). Thus, a CTGF promoter fragment between
805 and
+17 responds to TGF-
in a fibroblast-selective fashion.
TGF-
Induction of CTGF Promoter Activity in Fibroblasts Involves
Protein Kinase C and Ras/MEK/ERK and Is
Suppressed by MEKK1/JNK/c-Jun--
We decided to
examine known signaling pathways for their ability to modify the
TGF-
induction of CTGF in fibroblasts. For this analysis, we
transfected our full-length CTGF promoter/SEAP reporter construct into
fibroblasts. After an 18-h serum starvation step, cells were exposed
for 45 min to commercially available signal transduction pathway
inhibitors. Cells were then incubated for an additional 24 h
with or without 25 ng/ml TGF-
2. Preincubation of fibroblasts with
the protein kinase C (PKC)
,
,
,
, and
inhibitor Go
6850 (5 µM; bisindolylmaleimide, GF 109203X; Ref. 24)
blocked the TGF-
induction of CTGF promoter activity (Fig. 2, CTGF-SEAP).
Smad-dependent TGF-
induction of gene expression seemed
not to generally require PKC as Go 6850 did not block the TGF-
2-mediated induction of SBE-luciferase (Fig. 2,
SBE-LUX).

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Fig. 2.
Effect of signal transduction inhibitors on
TGF- 2-induced CTGF promoter activity in
fibroblasts. Inhibition of protein kinase C and Ras/MEK/ERK blocks
the TGF- induction of the CTGF promoter but not the generic
Smad-responsive promoter SBE-lux. PKC , , , , and inhibitor Go 6850 (5 µM; bisindolylmaleimide, GF 109203X;
Ref. 24) blocks the TGF- induction of CTGF promoter activity. PKC
and inhibitor Go 6976 (10 µM; Ref. 25) does not
affect the TGF- 2-mediated induction of CTGF. PKC , , , ,
and inhibitor Go 6983 (10 µM; Ref. 26) blocks the
TGF- induction of CTGF. A 10 µM concentration of the
MEK inhibitor U0126 (28) blocks the TGF- 2 induction of CTGF (this
figure and Ref. 15). A 20 µM concentration of the p38
inhibitor SB203580 (30) or a 10 µM concentration of the
JNK inhibitor SP600125 (31) has no impact on the TGF- 2 induction of
CTGF promoter activity in fibroblasts. The PKC and Ras/MEK/ERK cascades
do not generally affect Smad-dependent TGF- signaling as
the TGF- induction of the Smad-responsive reporter SBE-lux was not
affected by Go 6850 or U0126. NIH 3T3 cells were transfected,
processed, and analyzed as in Fig. 1. To assess the effect of
inhibitors on the TGF- induction of CTGF, cells were preincubated
for 45 min before an additional 24-h incubation with or without
TGF- 2. Expression values represent averages (±S.D.,
n = 6). **, statistically significant difference in
expression values (p < 0.05) relative to
controls.
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To further analyze the PKC dependence of the TGF-
induction of CTGF
in fibroblasts, we found that the PKC
and
inhibitor Go 6976 (10 µM; Ref. 25) did not affect the TGF-
2-mediated induction of CTGF (Fig. 2). Conversely the PKC
,
,
,
, and
inhibitor Go 6983 (10 µM; Ref. 26) blocked the
TGF-
induction of CTGF (Fig. 2). Thus, by comparing the selectivity
of the different PKC inhibitors used, we deduced that PKC
or
either alone or in combination is involved in the TGF-
induction of
CTGF in fibroblasts.
In many systems, MAP kinase pathways are involved in the TGF-
induction of gene expression. For example, TGF-
induces JNK and
Ras/MEK/ERK kinase pathways in fibroblasts (Refs. 15 and 27 and not
shown). We then investigated the role of MAP kinase pathways, namely
the Ras/MEK/ERK, p38, and JNK cascades, in the TGF-
induction of
CTGF. We found that a 10 µM concentration of the MEK 1 and 2 inhibitor U0126 (28) blocked the TGF-
2 induction of CTGF (Fig.
2; Ref. 15). Similarly dominant negative Ras (N17; Ref. 29) blocked the
TGF-
2 induction of CTGF promoter activity (Fig.
3, CTGF-SEAP). As for PKC, the
Ras/MEK/ERK cascade did not seem to directly affect the ability of
Smads to activate target gene expression as the TGF-
induction of
the Smad-dependent reporter SBE-lux was not affected by
either U0126 or dominant negative Ras (Figs. 2 and 3,
SBE-LUX).

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Fig. 3.
Effect of overexpressing wild-type or
dominant negative signal transduction components on
TGF- 2 induction of CTGF promoter activity in
fibroblasts. NIH 3T3 fibroblasts were transfected and assayed for
CTGF promoter activity as in Fig. 1B. The full-length CTGF
promoter/SEAP reporter construct or SBE-lux (0.5 µg/well) was
cotransfected with empty expression vector or expression vector
encoding dominant negative Ras (dnRAS), dominant negative
MKK4 (dnMKK4), dominant negative MEKK1 (dnMEKK1),
dominant negative c-Jun (TAM67, dnc-jun), wild-type c-Jun
(c-jun), or c-Jun with Smad3 and 4 (c-jun + Smad3/4; each at 0.5 µg/well) as indicated. All
experiments were performed in six-well plates. Cells were cotransfected
with a CMV promoter-driven vector encoding galactosidase (0.25 µg) to control for differences in transfection efficiency. Values
expressed are averages ± S.D. (n = 6). The effect
of overexpressing c-Jun was rescued by overexpressing Smad 3 and 4 (0.5 µg/well). Also, overexpressing c-Jun blocked the
Smad-dependent TGF- induction of SBE-lux suggesting that
c-Jun generally antagonizes Smad-dependent signaling. **,
statistically significant difference in expression values
(p < 0.05) relative to controls.
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We then continued our examination of MAP kinase signaling cascades in
CTGF gene regulation. We found that a 20 µM concentration of the p38 inhibitor SB203580 (30) or a 10 µM
concentration of the JNK inhibitor SP600125 (31) had no impact on the
TGF-
2 induction of CTGF promoter activity in fibroblasts (Fig. 2).
Similarly dominant negative MKK4 (an activator of p38 and JNK; Ref. 32) had no impact on the TGF-
2 induction of CTGF promoter activity (Fig.
3). However, overexpressing the JNK cascade mediator MEKK1 (33) blocked
CTGF induction (Fig. 3). Furthermore, overexpressing the JNK target
c-Jun reduced the TGF-
induction of the CTGF promoter (Fig. 3).
Similarly overexpressing dominant negative c-Jun (TAM67, which lacks
the activation domain of c-Jun; Ref. 34) increased TGF-
-induced CTGF
promoter activity.
We then assessed whether the ability of c-Jun to suppress the TGF-
induction of CTGF promoter activity could be via a
Smad-dependent mechanism. First, we found that the ability
of overexpressed c-Jun to suppress TGF-
-induced CTGF promoter
activity was rescued by overexpressing Smad 3 and 4 (Fig. 3,
CTGF-SEAP). Second, we found that c-Jun seemed to generally
suppress Smad-dependent activation of target gene
expression as overexpressing c-Jun markedly attenuated the ability of
TGF-
to induce SBE-lux (Fig. 3, SBE-LUX). These results
are consistent with the notion that activated c-Jun might suppress
Smad-dependent gene activation by sequestering Smads and
preventing them from activating gene expression of
non-Ap-1-dependent promoters (see "Discussion"). In
summary, our studies investigating the role of MAP kinase pathways in
CTGF gene expression implied that the MAP kinase pathway Ras/MEK/ERK is
necessary for the TGF-
induction of CTGF promoter expression in
fibroblasts and that the JNK MAP kinase cascade is refractory to this
process. Therefore, a proper balance between activation of the
Ras/MEK/ERK MAP kinase cascade relative to the JNK MAP kinase cascade
is important in controlling CTGF induction in fibroblasts.
An Element in the CTGF Promoter Is Sufficient to Confer
Fibroblast-specific Responsiveness to a Heterologous
Promoter--
To further characterize the element in the CTGF
promoter necessary for its fibroblast-specific induction by TGF-
, we
sought to identify elements in the CTGF promoter sufficient to confer fibroblast-specific TGF-
responsiveness to a heterologous promoter. We subcloned a fragment of the CTGF promoter between
805 and
23
upstream of the minimal, non-TGF-
-responsive herpes simplex virus TK
promoter (21). Confirming previous results (21), we found that, in
fibroblasts, the segment of the CTGF promoter lying between
805 and
23 conferred TGF-
responsiveness to the TK promoter (Fig.
4A). However, this promoter
construct did not confer TGF-
responsiveness to the TK promoter in
MvLu epithelial cells (Fig. 4A). Thus, the fragment of the
CTGF promoter that lies between nucleotides
805 and
23 is
sufficient to confer fibroblast-specific TGF-
responsiveness to a
heterologous promoter.

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Fig. 4.
Delineation of the CTGF promoter elements
necessary and sufficient to confer TGF-
responsiveness in fibroblasts to a heterologous (herpes simplex
virus minimal TK) promoter. A, elements between
nucleotides 244 and 143 and 126 and 77 are necessary for the
TGF- response (p < 0.05). Various CTGF promoter
fragments were subcloned into pTK-SEAP (Clontech)
upstream of the minimal TK promoter. The resultant constructs were
transfected into cells as indicated along with a CMV- -galactosidase
control plasmid. After a serum starvation step of 18 h, cells were
incubated with and without 25 ng/ml TGF- 2 for 24 h. Previously
the region of the CTGF promoter between 244 and 143 was analyzed
for its role in TGF- induction of CTGF promoter activity and was
found to consist of a functional Smad element (20). B,
mapping of the TGF- -responsive element between nucleotides 126 and
77 of the CTGF promoter. The TK/SEAP promoter/reporter construct
driven by nucleotides 351 to 77 of the CTGF promoter was mutated
between nucleotides 126 and 77 as indicated. Five linker scanning
CTGF promoter mutants were generated (m1-m5). The resultant
constructs were transfected into NIH 3T3 fibroblasts and incubated with
or without TGF- as in part A. Mutation of nucleotides
97 to 92 or 92 to 83 (m3 or m4) abolished
the ability of the CTGF promoter fragment to confer TGF-
responsiveness in fibroblasts to the TK promoter (p < 0.05). Values expressed are averages of adjusted SEAP expression ± S.D. (n = 6). ND, not done;
HFF, human foreskin fibroblasts.
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To precisely identify the CTGF promoter elements required for
fibroblast-selective TGF-
induction, we introduced a progressive series of 5' and 3' deletions in the CTGF promoter and assayed the
ability of these fragments to confer TGF-
responsiveness to the TK
promoter. CTGF/TK promoter constructs that lacked either nucleotides
244 to
143 or
126 to
77 of the CTGF promoter no longer
responded to TGF-
in fibroblasts (Fig. 4A). The element in the CTGF promoter between nucleotides
244 and
143 has been extensively analyzed. This CTGF promoter segment consists of two elements involved with CTGF gene expression: the BCE-1 site necessary for basal CTGF promoter activity in fibroblasts (20) and a Smad site
necessary for the TGF-
response (Ref. 20 and Fig.
5). Accordingly, for further study, we
focused on analyzing the contribution of the region of the CTGF
promoter between
126 and
77 to the induction of CTGF by
TGF-
2.

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Fig. 5.
The sequence 5'-GAGGAATG-3' is necessary for
the TGF- induction of CTGF in
fibroblasts. A full-length CTGF promoter (spanning nucleotides
805 to +17)/SEAP reporter construct ( 805) or otherwise identical
constructs possessing either a scrambled 5'-GAGGAATG motif
(5'), a scrambled 3'-GAGGAATG motif (3'), both
GAGGAATG motifs scrambled (BOTH), or a mutated Smad motif
(SMAD; Ref. 20) were transfected into NIH 3T3 cells. After
an 18-h incubation in serum-free media, TGF- 2 (25 ng/ml) was
added for a further 24 h. All experiments were performed in
six-well plates. Cells were cotransfected with a CMV promoter-driven
vector encoding -galactosidase (0.25 µg) to control for
differences in transfection efficiency. Values expressed are averages
of adjusted SEAP expression ± S.D. (n = 6). **,
statistically significant difference in expression values
(p < 0.05) relative to controls.
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A Repeat of the Sequence 5'-GAGGAATG-3', Located between
126 and
77 of the CTGF Promoter, Is Necessary for the Fibroblast-specific
Induction of CTGF by TGF-
--
Nucleotides
351 to
77 of the
CTGF promoter conferred TGF-
responsiveness in fibroblasts to the
minimal TK promoter (Fig. 4A). However, nucleotides
351 to
126 were not able to confer TGF-
inducibility to the TK promoter
(Fig. 4A). To further characterize the contribution of
nucleotides
126 to
77 to TGF-
induction of the CTGF
promoter, we decided to mutate the
351 to
77 CTGF promoter fragment
between nucleotides
126 and
77 and assess the ability of the
resultant mutant CTGF promoter fragments to confer TGF-
responsiveness to the TK promoter. The sequence between nucleotides
126 and
77 of the resultant CTGF promoter mutants is shown in Fig.
4B. We found that although three of the new mutant CTGF/TK
promoter constructs could still respond to TGF-
, constructs bearing
mutations that disrupted either nucleotides
97 to
92 or
92 to
83 no longer responded to TGF-
treatment (Fig. 4B, m3 and m4). Interestingly these regions of the
CTGF promoter each contain a single copy of the nucleotides
5'-GAGGAATG-3' (Fig. 5, underlined). Mutation of either the
5' or the 3' repeat or both repeats (Fig. 5, 5',
3', and BOTH) in the context of a full-length CTGF promoter (
805 to +17)/SEAP reporter construct abolished the
ability of the CTGF promoter to respond to TGF-
in fibroblasts (Fig.
5). This result was similar to the effect of mutating the Smad element
in the context of the
805 construct (Fig. 5, SMAD). Thus
both the Smad and the GAGGAATG motifs are necessary for the fibroblast-selective TGF-
induction of the CTGF promoter.
Nuclear Factors Greatly Enriched in Fibroblasts, Relative to
Epithelial Cells, Bind the GAGGAATG TGF-
Response Element of the
CTGF Promoter--
To determine whether proteins from fibroblast
nuclear extracts specifically bound the GAGGAATG motifs in the CTGF
promoter, we subjected a radiolabeled double-stranded oligomer spanning nucleotides
126 to
77 of the CTGF promoter to gel shift
analysis with nuclear extracts prepared from dermal fibroblasts. One
protein-DNA complex formed when we combined probe and fibroblast
nuclear protein (Fig. 6). This complex
was specific as formation of this complex was inhibited by adding a
100-fold molar excess of unlabeled wild-type probe (Fig. 6,
wt) but not by otherwise identical oligomers containing mutations in either the 5' or 3' GAGGAATG repeat (Fig. 6, 5'
and 3'). Conversely protein from nuclear extracts of MvLu
epithelial cells did not appreciably bind our
126 to
77 CTGF
promoter probe (Fig. 6). Thus, binding of fibroblast-enriched nuclear
factors to the GAGGAATG element of the CTGF promoter seemed to be in
part responsible for the fibroblast-specific induction of the CTGF promoter by TGF-
2.

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Fig. 6.
Gel shift analysis of the CTGF promoter
between nucleotides 126 and 77 reveal greatly enriched binding of
fibroblast, relative to epithelia, nuclear proteins. A
double-stranded DNA oligomer spanning nucleotides 126 to 77 of the
CTGF promoter was end-labeled and subjected to gel shift analysis with
nuclear extracts from human foreskin fibroblast (hff) or
MvLu cells as indicated (5 µg/lane). Competitors used (200-fold molar
excess) were cold wild-type probe (wt) or oligomers
identical to the probe but possessing mutations in either the 5'- or
3'-GAGGAATG motifs as indicated.
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The precise identity of these fibroblast factors binding the GAGGAATG
TGF-
response element of the CTGF promoter is under investigation;
however, it did not escape our notice that the sequence GAGGAATG is a
perfect consensus binding motif for the TEF/TEAD family of
transcription factors (35-39). To assess whether members of this
family could modify the TGF-
induction of CTGF in fibroblasts, we
cotransfected into fibroblasts empty expression vector
(EMPTY) or expression vector encoding the ubiquitous,
prototypical member of the TEF/TEAD family, TEF-1, along with the
full-length (
805 to +17) CTGF promoter/SEAP reporter construct.
Overexpression of TEF-1 suppressed basal and TGF-
-induced CTGF
promoter activity (Fig. 7,
CTGF-SEAP). Conversely overexpression of TEF-1 had no effect
on the TGF-
induction of SBE-lux (Fig. 7, SBE-lux). It is
possible that overexpression of TEF-1 competed for the binding to the
CTGF promoter of a TEF/TEAD family member that is required for basal
and TGF-
-induced gene expression. Interestingly this phenomenon of
squelching by the overexpression of TEF-1 is a general characteristic
of TEF-regulated genes; that is, overexpression of TEF-1 generally
suppresses TEF-responsive promoters (38). In the literature, these
results have been interpreted by hypothesizing that overexpression of
TEF-1 results in the sequestering of a limiting, common cofactor
necessary for the ability of TEF family members to induce expression of
target promoters (38, 39). Confirming this notion, overexpression of
the TEF family cofactor YAP65 (40) markedly attenuated the ability of
transfected TEF-1 to suppress the induction of the CTGF promoter by
TGF-
(Fig. 8). Furthermore,
overexpression of YAP65 increased basal and TGF-
-induced CTGF
promoter activity, suggesting that the expression of CTGF might be
dependent on the YAP65 transcriptional coactivator.

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Fig. 7.
Transfection of expression vector encoding
TEF-1 reduces basal and TGF- -induced CTGF
expression. NIH 3T3 cells were transfected with a full-length CTGF
promoter ( 805 to +17)/SEAP reporter plasmid or SBE-lux (0.5 µg/well) along with either empty expression vector or expression
vector encoding TEF-1 as indicated (1.0 µg/well). After an 18-h
incubation in serum-free media, TGF- 2 (25 ng/ml) was added
for a further 24 h. All experiments were performed in six-well
plates. Cells were cotransfected with a CMV promoter-driven vector
encoding -galactosidase (0.25 µg) to control for differences in
transfection efficiency. Values expressed are averages of adjusted SEAP
expression ± S.D. (n = 6). **, statistically
significant difference in expression values (p < 0.05)
relative to controls.
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Fig. 8.
The TEF coactivator YAP65 activates basal and
TGF- -induced CTGF promoter activity. NIH
3T3 cells were transfected with a full-length CTGF promoter ( 805 to
+17)/SEAP reporter plasmid (0.5 µg/well) along with either empty
expression vector or expression vectors encoding TEF-1 or YAP65 as
indicated (0.5 µg each/well). After an 18-h incubation in serum-free
media, TGF- 2 (25 ng/ml) was added for a further 24 h.
All experiments were performed in six-well plates. Cells were
cotransfected with a CMV promoter-driven vector encoding galactosidase (0.25 µg) to control for differences in transfection
efficiency. Values expressed are averages of adjusted SEAP
expression ± S.D. (n = 6). **, statistically
significant difference in expression values (p < 0.05)
relative to controls.
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That overexpression of TEF-1 reduced basal CTGF promoter activity and
blocked the TGF-
induction of CTGF and that factor binding to the
consensus TEF binding element, GGAATGG, of the CTGF promoter was
greatly enriched in fibroblasts relative to epithelial cells suggests
that a fibroblast-enriched member of the TEF/TEAD family of
transcription factors, or at least factors that compete with binding of
TEF/TEAD family members to this element, may act with the Ras/MEK/ERK
cascade and Smads to permit the fibroblast-specific induction of CTGF
by TGF-
2.
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DISCUSSION |
Our interest in examining CTGF expression arises out of a desire
to block the expression or activity of a downstream mediator of the
profibrotic effects of TGF-
but to leave other effects of TGF-
intact. CTGF is induced by TGF-
in fibroblasts and not in
keratinocytes (17) and acts with TGF-
to promote sustained fibrosis
(12). Thus, CTGF is likely to be a fibroblast-selective effector of the
profibrotic effects of TGF-
. We believe that understanding the
regulation of CTGF in fibroblasts cells should therefore provide novel
methods of developing antifibrotic therapeutics and that these
therapies would be perhaps more selective than generally blocking
TGF-
signaling (16). Intriguingly a small molecule inhibitor of CTGF
expression, namely Iloprost, has recently been shown to alleviate
fibrosis in scleroderma patients (14, 15).
In addition to the general Smad pathway, TGF-
activates other
signaling pathways, the nature of which depends on the cell type or
target of interest (41). The ability of TGF-
to specifically activate target genes is considered to be due in part to the
interaction of the general, ubiquitous TGF-
-stimulated Smad
signaling pathway with these other signaling pathways. For
example, in addition to the effects on Smad phosphorylation, TGF-
can signal via Ras and Rac proteins and activate certain MAP kinases
including the extracellular signal-regulated kinases ERK 1 and 2 and
JNK (42-44). In this report, we have found that Ras/MEK/ERK
potentiates but that JNK suppresses TGF-
-induced CTGF gene
expression in fibroblasts. Targeting the TGF-
induction of
Ras/MEK/ERK in fibroblasts may be useful in developing novel
antifibrotic therapeutics.
The antagonistic nature of Ras/MEK/ERK and JNK in the induction of CTGF
is intriguing. The TGF-
induction of JNK seems to be ubiquitous
(45-47). Similarly the TGF-
induction of Ras/MEK/ERK appears to be
ubiquitous, for example, occurring in cells lines known to induce CTGF
in response to TGF-
treatment, including fibroblast and mesangial
cells (15, 48), and in cells that do not induce CTGF such as epithelial
cells (41). However, Ras/MEK/ERK seems to modulate
TGF-
-dependent transcription in a promoter-specific fashion as blocking this cascade did not affect the ability of TGF-
to induce expression of the generic Smad-responsive promoter SBE-lux
(this report). This interpretation that Ras/MEK/ERK seems not to be
directly involved with Smad action is consistent with our recent
observations that antagonizing Ras/MEK/ERK does not significantly
affect Smad phosphorylation or the ability of Smad 3/4 to activate
expression of target promoters such as CTGF (15, 22).
In physiologically relevant promoters, Smads act with other
transcription factors, the identity of which depends on the gene of
interest, to induce promoter activity. In some promoters, Smads act
with Ap-1/c-Jun to induce gene expression; and in these cases, activating JNK/c-Jun enhances expression of target genes (49-53). However, in promoters whose induction does not involve Ap-1,
overexpressing c-Jun or JNK or activating JNK inhibits TGF-
-induced
expression (Refs. 54 and 55 and this report). These opposing effects may be due to competition for limiting amounts of Smads (54, 55) as the
effect of overexpressing c-Jun on the TGF-
induction of CTGF can be
rescued by overexpressing Smad 3/4 (this report). Thus, the TGF-
induction of Ras/MEK/ERK seems to be profibrotic, that is,
necessary for TGF-
-induced fibrosis in vivo (15). Conversely activation of JNK would be expected to be antifibrotic as
this pathway suppresses the induction of both collagen and CTGF by
TGF-
(Refs. 54 and 55 and this report). Thus which MAP kinase
pathway is predominantly active in fibroblasts at a given juncture is
likely to have a profound influence on the successful termination of
wound healing or on the perpetuation of the fibrotic response.
Therefore, pharmacological alteration of MAP kinase pathways in
fibroblasts may be useful in developing novel antifibrotic therapies.
In physiologically relevant promoters, Smads are thought not themselves
to activate gene expression but are believed to act with basal
transcription factors, which vary depending on the gene of interest, to
activate gene expression. The finding that a consensus TEF/TEAD
binding element was essential for the TGF-
induction of CTGF is
intriguing because to our knowledge these elements have not been
implicated in the control of TGF-
-induced gene expression. The
TEF/TEAD family possesses at least four members that are expressed in a
tissue- and developmental stage-specific fashion and have been
shown to be involved with activation of tissue-specific gene expression
(35-39, 56-59). However, recently an additional TEF family member was
discovered that was induced in fibroblasts by fibroblast growth factor
and serum (60). In addition, splice variants of known TEF family
members have been found to have different sequence binding or
activation specificity (60, 61), and there may be cell type-specific
methods of activating these transcription factors (59).
Taken together with our observation that factors present in fibroblast,
but not epithelial, nuclear extracts bound a region of the CTGF
promoter that was required for the fibroblast-specific induction of
CTGF by TGF-
, these observations raise the intriguing possibility
that novel factors of the TEF family might yet be found that are
involved with the activation of CTGF expression in fibroblasts.
In conclusion, as far as we are aware this is the first
report characterizing a TGF-
-responsive promoter element that acts in fibroblasts but not in epithelial cells. We have identified a
central role for protein kinase C, Ras/MEK/ERK, and a consensus TEF/TEAD binding motif in the fibroblast-selective and
promoter-specific induction of CTGF by TGF-
. Specifically how these
components interact to activate CTGF expression awaits further
characterization and cloning of the factor binding to the consensus TEF
motif in the CTGF promoter. Given the role of CTGF in sustained
fibrosis, identifying signaling pathways, trans-acting factors, and
cis-acting sequences involved with the TGF-
-specific induction
of CTGF in fibroblasts should continue to suggest new targets around
which to develop new, selective antifibrotic strategies.