From the Center for Rheumatology, Royal Free and
University College Medical School, Rowland Hill Street, London NW3
2PF, United Kingdom and ¶ FibroGen, Inc., South San Francisco,
California 94080
Received for publication, November 7, 2000, and in revised form, December 11, 2000
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ABSTRACT |
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In normal adult fibroblasts, transforming growth
factor- Wound healing requires the synthesis and reconstitution of
properly organized connective tissue. If activation of collagen gene
expression persists, uncontrolled connective tissue deposition results,
leading to pathologic scarring and fibrosis (1-3) such as in
scleroderma (systemic sclerosis), which is characterized by the
progressive scarring of skin and certain internal organs (4). Given the
ability of TGF In terms of scleroderma, the data supporting the role of TGF In an initial attempt to molecularly characterize the scleroderma
phenotype, we recently used differential display analysis to identify
genes up-regulated in dermal fibroblasts cultured from patients with
scleroderma (12). Perhaps the most interesting gene up-regulated in
scleroderma fibroblasts was connective tissue growth factor
(CTGF) (12). CTGF is a heparin-binding 38-kDa cysteine-rich
peptide that induces proliferation, collagen synthesis, and chemotaxis
in mesenchymal cells (13-18) and has been shown to potentiate
sustained fibrosis when injected along with TGF In contrast to the situation in fibrotic disorders, CTGF is not
expressed in normal human dermal or mouse NIH 3T3 fibroblasts unless
cells are treated with TGF Activation of TGF In this report, we assess the role of SMADs in CTGF gene
expression in normal and scleroderma fibroblasts. Using a combination of Western blot, gel shift, and gene cotransfection/promoter assays, we
show that a functional SMAD binding site in the CTGF
promoter is necessary for the induction of CTGF by TGF Cell Culture, Reporter Assays, and Transfections--
NIH 3T3
fibroblasts (ATCC) were maintained in DMEM, 10% calf serum (Life
Technologies). Fetal SMAD3-knockout mouse
fibroblasts, and their wild-type counterparts, were a gift from Anita
Roberts (National Institutes of Health). Primary dermal fibroblasts
from lesional areas of scleroderma patients and normal individuals were
taken from biopsies of patients with diffuse cutaneous scleroderma and
age, sex, and anatomically site-matched healthy volunteers, respectively. All patients fulfilled the criteria of the American College of Rheumatology for the diagnosis of scleroderma. Informed consent and ethical approval were obtained for all procedures. Fibroblasts were maintained in DMEM supplemented with 10% fetal calf
serum, 100 units/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine (Life Technologies). All cells
were used between passages 2 and 5, with reculture at a dilution of 1 in 3 passaged fibroblasts (41). Transfections were performed in 6-well
plates using LipofectAMINE plus, as described by the manufacturer (Life Technologies). For reporter assays, 1 µg of a reporter construct was
cotransfected with 0.25 µg of CMV DNA Constructs--
The full-length CTGF
promoter/SEAP reporter construct is described elsewhere (20). Point
mutations were introduced using a kit (Stratagene). The mutagenic
primer (Sigma Genosys) for the SMAD mutant was
5'-GAGCTGGAGTGTGCCAGCTTTTGGATCCGGAGGAATGCTGAGTGTC-3' and for the
TGF Western Blot Analysis--
Human dermal fibroblasts were grown
in DMEM and 10% fetal bovine serum. Cells were grown to confluence and
switched to DMEM supplemented with insulin/transferrin/selenium. Cells
were grown for an additional 18 h, and then 10 ng/ml TGF Electrophoretic Mobility Shift Assays--
Protein extracts were
prepared from ~1 × 106 NIH 3T3 control cells or
cells treated with TGF Immunoprecipitations--
NIH 3T3 cells were transfected as
described above with 2 µg each of expression vector encoding
SMAD3·FLAG, SMAD4·FLAG, ski·HA or snoN·HA per 100-mm dish.
Forty-eight hours later, cells were lysed in 800 µl of ice-cold
freshly prepared 50 mM Hepes, 10% glycerol, 0.5% Nonidet
P-40, 50 mM NaCl, 1 mM dithiothreitol, 50 mM sodium fluoride, 25 mM SMADs Are Necessary for the TGF
To determine whether SMAD proteins could bind to the putative SMAD
binding site, we performed a gel shift assay with NIH 3T3 fibroblast
nuclear extract, made from cells that had been treated with TGF
To determine whether SMADs could regulate CTGF gene
expression, we cotransfected expression vectors encoding various SMADs with a full-length CTGF promoter/SEAP construct into NIH 3T3
fibroblasts. Cotransfection of SMAD3 and SMAD4 enhanced CTGF expression
significantly (Fig. 3A).
However, cotransfection of SMAD2 and SMAD4 did not potentiate the
TGF
Our transfection data suggested that SMAD3 is principally involved in
the TGF Regulators of SMADs Modulate TGF
To verify that ski and snoN could interact with SMADs in NIH 3T3
fibroblasts, we cotransfected expression vectors encoding FLAG-tagged
SMADs 3 and 4 along with expression vectors encoding either HA-tagged
ski or HA-tagged snoN into NIH 3T3 fibroblasts. The presence of these
molecular tags allowed easy recognition and manipulation of the
transfected proteins. After transfection, cells were serum-starved for
24 h and were then lysed. Because 20 min of TGF
To determine whether HA-tagged ski and snoN could bind to FLAG-tagged
SMADs to inactivate them, we then immunoprecipitated lysates with an
anti-FLAG antibody. In extracts from cells that had been cotransfected
with HA-tagged ski and FLAG-tagged SMAD3 and SMAD4, we able to
immunoprecipitate HA-tagged ski with anti-FLAG-agarose gel, as seen
with an anti-HA antibody (Fig. 5A, IP-FLAG).
Conversely, no HA-tagged ski was immunoprecipitated when unconjugated
beads were used (Fig. 5A, IP-BEADS). Similarly,
in extracts from cells transfected with FLAG-tagged SMADs 3 and 4 and
HA-tagged snoN, we were able to immunoprecipitate snoN with
anti-FLAG-agarose gel (Fig. 5, IP-FLAG) but not with
unconjugated agarose (Fig. 5A, IP-BEADS). We
found that addition of TGF
We then assessed the effect of transfected ski and snoN constructs on
CTGF promoter activity in NIH 3T3 fibroblasts when
visualized by readout from a full-length CTGF promoter/SEAP
reporter construct. We found that ski and snoN suppressed the induction
of CTGF promoter activity by TGF CTGF Expression in Scleroderma Fibroblasts Is Independent of SMAD
Signaling--
Previously, we showed that CTGF protein was
constitutively expressed in fibroblasts cultured from lesional areas of
scleroderma patients, even in the absence of exogenous TGF
To further clarify the potential role of SMADs in the scleroderma
phenotype, we used Western blot analysis to compare the levels of
expression of SMADs 3, 4, and 7 (because of their role in
CTGF gene expression) in normal and scleroderma dermal
fibroblasts. We used dermal fibroblasts cultured from three normal
individuals and affected areas of three individuals with scleroderma.
The scleroderma fibroblasts chosen had the highest levels of
CTGF promoter activity. Bands were quantified relative to a
vimentin standard (Fig. 6B). Although some individuals
showed elevated levels of particular SMAD proteins, no overall picture
emerged which correlated SMAD levels with the scleroderma phenotype;
for example individual S3 had the highest SMAD3 level but also had extremely high SMAD7 (Fig. 6B). That is, our results suggest
that the elevated level of CTGF expression observed in scleroderma appears to be independent of SMAD action.
To further clarify if elevated SMAD signaling was responsible for
activated expression of genes in scleroderma, we transfected into
scleroderma and normal fibroblasts a construct containing the human
plasminogen activator inhibitor (PAI-1) promoter linked to
firefly luciferase. This construct has been previously shown to be
responsive to SMAD-dependent TGF
To determine whether the elevated level of CTGF promoter
activity in scleroderma cells reflected elevated level of basal
promoter activity, we transfected the TGF TGF In adults, constitutive CTGF expression is a consistent marker of the
fibrotic phenotype (12, 20-24). After we established the role of SMADs
in the TGF Collectively, these results suggest that the maintenance of the
scleroderma phenotype, as visualized by elevated CTGF levels, is not
caused by SMAD-dependent TGF These results are consistent with previous studies that have examined
the role of TGF (TGF
) induces the expression of connective tissue growth
factor (CTGF). CTGF independently promotes fibroblast
proliferation and matrix deposition, and in acute models of fibrosis
promotes cell proliferation and collagen deposition acting
synergistically with TGF
. In contrast to normal fibroblasts,
fibroblasts cultured from fibrotic tissues express high basal levels of
CTGF, even in the absence of added TGF
. Induction of transcription
by TGF
requires the action of SMAD proteins. In this report we have
investigated the role of SMADs in the TGF
-induction of CTGF in
normal fibroblasts and in the elevated levels of CTGF expression found
in dermal fibroblasts cultured from lesional areas of patients with
scleroderma, a progressive fibrotic disorder that can affect all organs
of the body. We have identified a functional SMAD binding site in the
CTGF promoter. TGF
-induction of CTGF
is dependent on SMAD3 and SMAD4 but not SMAD2 and is p300-independent.
However, mutation of the SMAD binding site does not reduce the high
level of CTGF promoter activity observed in dermal
fibroblasts cultured from lesional areas of scleroderma patients.
Conversely, the previously termed TGF
RE in the CTGF
promoter is required for basal CTGF promoter activity in
normal fibroblasts and for the elevated level of CTGF
promoter activity in scleroderma fibroblasts. Thus, the maintenance of
the fibrotic phenotype in scleroderma fibroblasts, as visualized by
excess CTGF expression, appears to be independent of
SMAD-dependent TGF
signaling. Furthermore, given
CTGF's activities, the high level of CTGF expression observed in
scleroderma lesions may contribute to the excessive scarring observed
in this disorder.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 to promote
fibroblast proliferation and matrix synthesis, attention has been
devoted to its potential role in initiating and maintaining the
fibrotic phenotype (for reviews, see Refs. 5 and 6), including
scleroderma (7). For example, there is a clear correlation between
TGF
action and the initiation of fibrosis; in acute drug- or
surgery-induced animal models, anti-TGF
strategies are effective at
blockading the onset of fibrosis (for review, see Ref. 6). However, the
fibrosis is clinically a chronic disorder; the involvement of TGF
in
the maintenance of fibrosis and the effectiveness of anti-TGF
strategies in the reversion of fibrosis is unclear.
in the
fibrotic phenotype is circumstantial, chiefly depending on the
histological distribution of TGF
mRNA and protein.
Unfortunately, the data are often contradictory. For example,
mononuclear cells taken from bronchial levage fluids of
scleroderma patients have elevated TGF
levels (8). However, in the
actual lesional areas of skin, TGF
mRNA is only localized to the
leading edge of the scleroderma lesion; i.e. to the region
of enhanced inflammatory response that is presumably involved with the
initiation of the fibrotic response (9). Furthermore, fibroblasts taken
from scleroderma lesions show elevated levels of collagen relative to
their normal counterparts, yet show little difference in their ability
to produce TGF
or in their ability to bind TGF
, nor do they show
enhanced sensitivity to TGF
treatment (10, 11). Thus, although there
seems to be circumstantial data to support the role of TGF
in the
onset of the scleroderma fibrotic phenotype, it is unclear as to its
precise role in initiating or maintaining the scleroderma phenotype.
in an acute animal
model (19). Previously, CTGF mRNA and protein were shown to be
constitutively expressed in numerous fibrotic disorders both in skin
and in internal organs, such as atherosclerosis and pulmonary and renal
fibrosis, and that this expression correlated with high collagen
synthesis (12, 20-24). Hence, in adult tissues, constitutive CTGF
expression is considered a faithful, clinical, molecular marker of
fibrosis. Furthermore, given its activity, CTGF is considered a
mediator of the fibrotic phenotype (12-24).
(14-16, 20, 25). Similarly, exogenous
addition of TGF
increases the amount of CTGF protein and promoter
activity produced by fibroblasts cultured from scleroderma lesions (12,
20). This induction by TGF
is cell-type specific, as it occurs in
connective tissue cells but not in epithelial cells or lymphocytes
(14-16). The regulation of CTGF expression by TGF
appears to be
controlled primarily at the level of transcription (20, 25).
Originally, the up-regulation of CTGF by TGF
was thought to be
solely dependent on a relatively small sequence present in the 5'
upstream region of the CTGF promoter (TGF
response element; TGF
RE) (25). This sequence does not resemble the TGF
response elements described in other genes, including the SMAD recognition sequence (25). However, recently we have shown that sequences immediately upstream of the previously identified TGF
RE are required for TGF
to induce CTGF expression (20). Similarly, the
high level of CTGF protein observed in scleroderma appears to be due,
at least in part, to gene transcription because the CTGF
promoter activity is substantially higher in scleroderma dermal
fibroblasts relative to normal dermal fibroblasts (12). However, the
precise mechanism underlying the control of CTGF gene
expression in normal and fibrotic fibroblasts remains unknown.
-dependent gene expression is commonly
mediated through SMADs 2, 3, and 4 (for reviews, see Refs. 25 and 26).
SMADs 2 and 3 are normally present in the cytosol. Once activated by
TGF
, SMADs 2 and 3 interact transiently with type I TGF-
receptor
kinase and become phosphorylated at their carboxyl terminus (27, 28).
SMAD2 and SMAD3 then form a heteromeric complex with SMAD4 (29, 30).
These complexes then translocate to the nucleus and activate expression
of target genes (25), in concert with other nuclear factors the
identity of which can vary depending on the promoter and cell type
(e.g. Refs. 31-34). Recent studies have recognized a
consensus DNA motif, GTCTAGAC that provides the binding site for the
SMAD3·SMAD4 complex (35). Homologs of this element have been
identified in the promoters of several TGF
-responsive genes
(e.g. Refs. 36-39). More recently, studies in
TGF
-responsive systems have also identified other SMAD family
members, such as SMADs 6 and 7, which represent a functionally distinct
class of SMADs that antagonize SMAD-receptor interactions (40).
in
fibroblasts. Conversely, the constitutive CTGF expression observed in
scleroderma appears to be independent of SMAD action. Thus, the
maintenance of the fibrotic phenotype in scleroderma, as visualized by
constitutive CTGF expression, appears to be SMAD-independent. In
addition, we show that the previously termed TGF
RE is required for
basal CTGF expression in normal fibroblasts and elevated CTGF
expression in scleroderma.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase plasmid, which was
used as an internal transfection control
(CLONTECH). For assays in which the role of
transacting proteins was to be tested, 0.5 µg of reporter, and 1 µg
of empty vector or expression vector encoding the protein of interest
were used. For transfections involving ski and snoN, amounts of DNA
used were as described in the text. SEAP and
-galactosidase
kits were used to perform reporter gene assays (Tropix). TGF
2 was
from R&D Systems or Celtrix and used at concentrations indicated in the
text. The data are presented as mean ± S.E., or representative of
experiments performed in at least triplicate in at least two separate
experiments. Statistical analysis was performed by the Student's
unpaired t test. p values less than 0.05 were
considered statistically significant.
RE mutant was 5'-CAGACGGAGGAATGCTGGGGATCCCGAGGATCAATCC-3'. Constructs were fully sequenced in both directions to confirm mutagenesis before use. SMADs 2, 3, 4, 6, and 7 expression vectors were
generous gifts from Joan Massague (Sloan-Kettering), and ski and snoN
expression vectors were generous gifts from Robert Weinberg (Whitehead
Institute). Expression vectors encoding wild-type and dominant-negative
p300 were from Upstate Biotechnology. The human plasminogen activator
inhibitor (PAI-1)/luciferase construct was a generous gift from Dan
Rifkin (New York University).
2 (R&D
Systems) was added. Media was removed 24 h later, and 25 µl was
electrophoresed through a 12% SDS-polyacrylamide gel (Novex) and
blotted to nitrocellulose (Bio-Rad). Filters were blocked overnight at
4 °C in 5% nonfat dry milk in Tris-buffered saline, 0.1% Tween 20. CTGF protein was detected by a 1 h incubation of a 1:1000 dilution
of rabbit anti-CTGF antibody in 1% milk, TBS, 0.1% Tween 20. For
anti-SMADs 2, 3, and 7 Westerns, cells were lysed in 2% SDS, and
protein concentration was determined (Bio-Rad). Equal amounts of
protein were electrophoresed as described above. For experiments
involving SMADs, SMAD proteins were detected using a 1:1000 dilution of antibody. SMADs 3, 4, and 7 antibodies were from Santa Cruz
Biotechnology. For experiments involving the detection of transfected
FLAG-tagged SMADs 3 and 4, 1:5000 dilution of anti-FLAG antibody
(Sigma) was used. For detection of HA-tagged transfected ski and snoN,
an anti-HA antibody (Roche Molecular Biochemicals) was used. In all cases, proteins were detected by 1:5000 dilution of an appropriate horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch or Roche Molecular Biochemicals). Proteins were then
detected by chemiluminescence (Pierce). Bands on Western blots were
quantified using a computer program (AlphaEase, Alpha Innotech), and
values were expressed in arbitrary densitometric units.
2 (2 ng/ml) for 60 min following serum
starvation for 24 h. Cells were then lysed, and protein nuclear
extracts were prepared as previously described (42). Protein
concentrations were determined using the Bio-Rad protein assay kit.
Oligonucleotide probes as indicated were 32P-labeled, gel
purified, and incubated with nuclear extracts for 30 min at 30°. Gel
shift conditions consisted of 1-3 µl of nuclear extract (containing
2-5 µg protein) and 0.5 ng probe (1-5 × 104 cpm)
in a final volume of 15 µl of 150 mM NaCl, 10 mM Tris-HCl, pH 7.5 and 50 µg per ml poly(dI-dC) (Roche
Molecular Biochemicals). For antibody treatment, nuclear extracts were
incubated with 2 µg of the indicated antibodies prior to addition of
probe. SMAD2 antibody was from Zymed Laboratories Inc.
and SMAD3 and SMAD4 antibodies were from Santa Cruz Biotechnology. For
competition experiments, 50- or 100-fold excess cold competitor (25 or
50 ng, respectively of unlabeled probe) was added to the reaction mixture before incubation when required. Complexes were resolved on 6%
nondenaturing acrylamide gels using 0.5× Tris-Borate-EDTA. Gels
were then dried, and complexes were visualized by autoradiography.
-glycerol
phosphate, 1 mM EDTA, 1 mM sodium pervanadate,
2 mM phenylmethylsulfonyl fluoride, pH 7.9. Cells were
lysed on ice, scraped, sonicated, and spun. To 100 µl of lysate, 40 µl of agarose affinity gel conjugated with an anti-FLAG antibody
(Sigma) was added. Gel without antibody was used as a control. Gel and
protein slurry was rotated on a shaker for 2 h at 4o.
Beads were washed in lysis buffer (1 ml) three times. Protein was
eluted with protein sample buffer (100 µl) and stored at
80o. Samples (25 µl) were then subjected to
polyacrylamide gel electrophoresis through a 4-20% gel (Novex), and
gels were blotted onto polyvinylidene difluoride membrane (Millipore).
After blocking membrane for 2 h at room temperature in
phosphate-buffered saline, 5% nonfat dry milk, 0.1% Tween 20, blots
were incubated overnight at 4 oC with a 1:2000 dilution of
anti-HA antibody (Covance). After extensive washing, proteins were
detected by a 1:5000 dilution of horseradish peroxidase-conjugated goat
anti-mouse antibody (Roche Molecular Biochemicals or Jackson
Immunoresearch). Antibodies were diluted in blocking buffer. A
chemiluminescent detection kit was used to observe proteins (Pierce).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mediated Induction
of CTGF in a p300-independent Fashion--
Recently, we identified a
segment of the CTGF promoter between nucleotides
244 and
166 that was required for the TGF
-mediated induction of CTGF (20).
Sequence inspection of the region of the CTGF promoter
between
244 and
166 resulted in the identification of a putative
consensus SMAD site (35-38) immediately upstream of
166 (Fig.
1A). Mutating this sequence to
a BamHI site in an otherwise wild-type, full-length
CTGF promoter/SEAP reporter construct resulted in complete
abolition of TGF
-induced gene expression when this construct was
transfected into NIH 3T3 fibroblasts (Fig. 1B). Thus, a
consensus SMAD binding site seems necessary for TGF
-induction of
CTGF. Previously, a sequence immediately downstream of this element was
identified as being necessary for maximal TGF
induction of CTGF, and
hence was called the TGF
RE (25). To compare the relative
contribution of the SMAD site and the TGF
RE in terms of the TGF
induction of CTGF, we mutated the TGF
RE to a BamHI site.
In contrast to mutating the SMAD site, mutating the TGF
RE caused a
marked decrease in basal CTGF expression, whereas still permitting a
response to TGF
(Fig. 1C). Thus, the factor(s) binding to
the TGF
RE are required for basal CTGF transcription and
hence for the maximal response of the CTGF promoter to
TGF
(25).
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Fig. 1.
Effect of mutating a putative SMAD binding
site on CTGF expression. A, sequence of the
CTGF promoter between nucleotides 183 and
150, in gray.
The sequence between
244 and
166 has also previously been shown to
be necessary for TGF
-induced CTGF expression (Ref. 20). A consensus
SMAD binding site located between
173 and
166 is shown
underlined and in black. The sequence in
gray was made into a double-stranded oligomer for use as a
gel shift probe (see Fig. 2). B and C, in the
context of a full-length (805 base pair) promoter construct, the SMAD
binding site (B) or TGF
RE (Ref. 25, C) was
mutated to a BamHI site, as described under "Materials and
Methods." The resultant constructs (SMAD or TGFbRE, respectively)
were transfected into NIH 3T3 fibroblasts, and expression was compared
with that of the unmutated wild-type full-length construct
(FL). Cells were serum-starved for 24 h, followed by an
additional 24-h incubation with or without addition of 25 ng/ml TGF
2
(tgfb or no tgfb, respectively). Assays were
performed as described under "Materials and Methods." Relative
light units based on SEAP expression (25 µl of conditioned media)
normalized to
-galactosidase activity (from a cotransfected
CMV-
-galactosidase plasmid) are shown (mean ± S.E.,
n = 18).
. A
radiolabeled double-stranded oligonucleotide probe containing the
putative SMAD binding site was used as probe (see Fig. 1 for sequence).
We found that protein complexes could bind the probe (Fig.
2, arrow). This binding was
specific as binding could be competed by adding excess unlabeled
oligomer (Fig. 2). Binding of one of the complexes could be abolished
by preincubating nuclear extract with an anti-SMAD3 or anti-SMAD4
antibody, but not with an anti-SMAD2 antibody (Fig. 2). Collectively,
these data suggest that the putative SMAD site identified as being
important for gene expression is a bona fide SMAD binding
site. The components of the other complex are under further
investigation.
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Fig. 2.
SMADs 3 and 4 bind the CTGF
promoter. A double-stranded oligomer probe (0.5 ng)
containing the putative SMAD binding site of the CTGF
promoter (shown in Fig. 1) was used in a gel shift with nuclear
extracts prepared from NIH 3T3 cells that had been treated for 1 h
with TGF 2. A complex was observed (arrow) that was
specific because 50 or 100-fold excess (25 ng and 50 ng, respectively)
of cold competitor oligonucleotide could compete for binding, as shown.
Anti-SMAD2, 3, or 4 (1 µl) antibodies were preincubated with nuclear
extract before addition of probe.
-mediated induction of CTGF (Fig. 3A). Individually, only SMAD3 and SMAD4 mildly increased CTGF promoter activity
in the absence of added TGF
, with SMAD4-inducing activity to
approximately the same level as SMAD2 and SMAD4 combined (not shown).
Transfection of the inhibitory SMAD, SMAD7, markedly attenuated the
ability of TGF
and SMAD3 and 4 to increase CTGF promoter
activity, but had little effect on basal expression (Fig.
3A). Conversely, transfecting the inhibitory SMAD, SMAD6,
had no effect on the ability of TGF
to induce CTGF
promoter activity (Fig. 3A). SMADs often require p300 as a
transcriptional cofactor (32, 43). We found that transfecting either
wild-type or dominant-negative p300 had no impact on the
TGF
-induction of CTGF (Fig. 3B). Thus, SMADs modulate CTGF gene expression in a p300-independent fashion.
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Fig. 3.
Effect of transfecting SMAD expression
vectors on CTGF promoter activity in NIH 3T3
fibroblasts. A, cotransfecting SMAD3 and SMAD4 potently
activates CTGF expression (SMAD 3/4). Cotransfecting SMAD2
and 4 were less effective at activating expression (SMAD
2/4). Transfecting SMAD7, but not SMAD6 blocked TGF -induced
gene expression. See Fig. 2 and "Materials and Methods" for
details. B, cotransfecting a construct encoding wild-type
p300 (p300) or dominant-negative p300 (dn p300)
had no statistically significant effect on TGF
-induced CTGF
expression. The full-length CTGF promoter/SEAP plasmid was
used (0.5 µg/well). An empty expression vector (EMPTY) was
used as a control. Expression vectors were used at 1 µg/well.
Treatments had no effect on an SV40 promoter/enhancer/SEAP construct
(CLONTECH) transfected in parallel (not shown).
TGF
2 was used at 25 ng/ml (tgfb). No tgf, no
TGF
2 added. Relative light units were based on SEAP expression (25 µl of conditioned media) normalized to
-galactosidase activity
(from a cotransfected CMV-
-galactosidase plasmid) are shown
(mean ± S.E., n = 18, except SMAD6 and 7, n = 6).
-induction of CTGF. To verify this, we obtained fibroblasts
cultured from SMAD3-knockout and wild-type fetal mice (45).
We treated cells with TGF
for 24 h and assayed their ability to
induce CTGF protein. Both wild-type and knockout embryonic mouse
fibroblasts displayed constitutive CTGF expression. This result is not
surprising, because CTGF protein is expressed constitutively in several
cell types during development (for review, see Ref. 13). We note an
elevated basal level of CTGF in the SMAD3-knockout cells,
perhaps because of a compensation for a lack of inducibility by TGF
.
However, although wild-type mouse fibroblasts responded to TGF
by
elevating CTGF levels, fibroblasts cultured from
SMAD3-knockout animals could not respond to TGF
(Fig.
4). Thus, the TGF
-mediated induction
of CTGF requires SMAD3.
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Fig. 4.
TGF does not induce
CTGF in embryonic fibroblasts cultured from
SMAD3-knockout mice. SMAD3-knockout
and normal mouse embryonic fibroblasts (Ref. 45) were cultured and
serum-starved for 24 h. Cells were then grown for an additional
24 h with and without addition of TGF
2 (10 ng/ml). Western
blots for CTGF were performed on the cell layer (25 µg) as described
under "Materials and Methods". TGF
induced CTGF in normal
fibroblasts but not in SMAD3-knockout fibroblasts.
-induced CTGF Gene Expression,
ski and snoN--
To verify that SMADs activate CTGF gene
expression, we then examined the ability of several known regulators of
SMAD activity to modulate CTGF gene expression. Recently,
the oncoproteins ski and snoN have been shown to suppress SMAD
signaling in keratinocytes by binding directly to SMADs and thus
preventing their participation in functional transcriptional complexes
(46-48). We decided to use this fact to further probe the notion that
TGF
-mediated induction of CTGF was SMAD-dependent and to
determine whether this silencing mechanism was functional in fibroblasts.
treatment
degrades snoN (47), we also wanted to ensure that transfected snoN
existed even in the presence of exogenous TGF
. Thus, we also
prepared lysates from cells that had been treated with TGF
for 20 min immediately after the serum-starvation treatment. To verify
expression of transfected FLAG-tagged SMADs, whole cell lysate was then
subjected to SDS-polyacrylamide gel electrophoresis. By Western blot
analysis, transfected FLAG-tagged SMADs were readily detected with an
anti-FLAG antibody in lysates from cells that has been cotransfected
with either ski and snoN transfected cells (Fig.
5A, LYSATE).
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Fig. 5.
ski and snoN block the TGF
induction of CTGF. A, ski and snoN bind to SMADs
in NIH 3T3 fibroblasts. Cells were transfected with expression vectors
encoding SMAD3 and SMAD4, which were both FLAG-tagged and either sno or
ski, which were both HA-tagged (2 µg of each expression vector per
100-mm dish). Cells were serum-starved for 24 h after
transfection, after which cells were immediately lysed or treated for
an additional 20 min with 25 ng/ml TGF
2, as indicated
(tgfb). Whole lysates (25 µg/lane) were subjected to
SDS-polyacrylamide gel electrophoresis and Western analysis with an
anti-FLAG antibody, to verify expression of SMADs 3 and 4 (LYSATE). Equal amounts of lysate were then either
immunoprecipitated with unconjugated agarose gel (IP-BEADS)
or FLAG-agarose gel (IP-FLAG). Gels were then subjected to
Western analysis with anti-HA antibody to detect transfected HA-tagged
ski and snoN, which were detected only when anti-FLAG conjugated beads
were used. B, ski and snoN block the TGF
- and
SMAD-mediated induction of CTGF. Transfecting expression vectors
encoding ski and snoN block the TGF
- and SMAD-mediated induction of
CTGF. Transfecting different amounts of ski and snoN expression vectors
(as shown) into NIH 3T3 fibroblasts block the ability of TGF
(25 ng/ml) to induce CTGF promoter activity. The full-length
CTGF promoter/SEAP plasmid was used. Empty expression vector
(empty) was used as a control at equal amounts to ski or
snoN expression vector. Relative light units based on SEAP expression
(25 µl of conditioned media) normalized to
-galactosidase activity
(from a cotransfected CMV-
-galactosidase plasmid) are shown
(mean ± S.E., n = 6).
to cells for 20 min before lysis had no
detectable effect on anti-FLAG precipitable ski or snoN levels (Fig.
5A; TGF
-added lanes, IP-FLAG).
Thus, transfected ski and snoN bound with SMAD3/4 in NIH 3T3
fibroblasts and thus could be used to test the idea that
CTGF gene induction by TGF
is
SMAD-dependent.
(Fig. 5B).
There appeared to be a greater effect of ski than snoN on CTGF
expression, presumably because TGF
treatment degrades snoN (47).
Given the known abilities of ski and snoN (46-48), these results
support the idea that SMADs modulate CTGF gene expression.
(12, 20)
and that this elevated expression was due at least in part to high levels of CTGF promoter activity (12). To investigate the
role of SMAD-dependent signaling in the elevated level of
CTGF characteristic of scleroderma, we transfected our full-length
CTGF promoter/SEAP reporter construct into normal dermal or
scleroderma fibroblasts and compared its expression level to that of
our CTGF promoter/SEAP reporter construct with a mutated
SMAD binding site. We used dermal fibroblasts cultured from four normal
individuals and ten individuals with diffuse scleroderma. Mutation of
the SMAD binding site had no statistically significant effect on the
basal level of promoter activity either in normal or scleroderma
fibroblasts (Fig. 6A; Student's unpaired t test, p > 0.05).
Thus, the high level of CTGF promoter activity observed in
scleroderma fibroblasts is not dependent on the SMAD recognition
sequence.
View larger version (22K):
[in a new window]
Fig. 6.
Contribution of SMADs to basal CTGF
expression in scleroderma fibroblasts. A, mutation of
the SMAD binding element does not reduce the high level of
CTGF promoter activity in scleroderma fibroblasts. Either
the full-length CTGF promoter/reporter construct
(FL) or the SMAD mutant promoter/reporter
construct (SMAD) was transfected into normal dermal
fibroblasts (from four individuals) or scleroderma fibroblasts (from
ten individuals). Each experiment was performed in triplicate and
normalized for transfection efficiency (with cotransfected CMV
-galactosidase plasmid). Values represent the average (± S.E.) of
all trials (i.e. n = 12 and
n = 30, respectively). B, SMADs 3, 4, and 7 levels in normal and scleroderma fibroblasts. Densitometric analysis
was preformed as described under "Materials and Methods." Values
shown are arbitrary densitometric units adjusted for vimentin levels in
each lane. Fibroblasts cultured from three normal individuals
(N1, N2, N3) and three individuals
with scleroderma with the highest CTGF promoter activity
(S1, S2, S3) are shown.
signaling (49, 50). No
elevation of PAI-1 promoter activity was observed in
scleroderma fibroblasts, suggesting that SMAD-dependent
TGF
signaling is not generally activated in scleroderma fibroblasts
(Fig. 7).
View larger version (15K):
[in a new window]
Fig. 7.
PAI-1 promoter activity is not
elevated in scleroderma fibroblasts. A construct containing the
SMAD-responsive 800-base pair PAI-1 promoter linker to the
luciferase reporter gene (Refs. 49, 50) was transfected into normal
(two individuals) and scleroderma fibroblasts (two individuals). Each
experiment was performed in triplicate (i.e.
n = 6) and normalized for transfection efficiency using
a cotransfected CMV- -galactosidase plasmid. Values reflect the
average (± S.E.) of all trials.
RE-mutant CTGF
promoter/SEAP reporter construct into normal and scleroderma
fibroblasts. We found that mutating the TGF
RE reduced
CTGF promoter levels in both normal and scleroderma cells
(Fig. 8). In fact, removing this element
reduced CTGF promoter levels observed in scleroderma fibroblasts to that of levels in normal fibroblasts, suggesting that
this element is involved with the high level of CTGF expression observed in scleroderma.
View larger version (32K):
[in a new window]
Fig. 8.
Mutation of the
TGF RE reduces CTGF promoter
activity in scleroderma fibroblasts. Experiments were performed
identical to those in Fig. 6, except the TGF
RE mutant
promoter/reporter construct (TGFbRE) was transfected into
normal and scleroderma dermal fibroblasts and compared with that of the
full-length promoter construct transfected into the same cells
(FL). Values represent the average (± S.E.) of all trials
(i.e. n = 12 and n = 30, respectively).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
is known to activate gene expression through the action of
SMAD proteins (25, 26). In the absence of TGF
, SMADs 2 and 3 are
primarily cytosolic. When TGF
is present, SMADs 2 and 3 are
phosphorylated by the TGF
receptor, bind to SMAD 4, and migrate into
the nucleus to activate expression of TGF
-responsive genes. The
inhibitory SMADs, SMAD6 and SMAD7, antagonize this pathway of signaling
by TGF
family members (39, 40). For the CTGF promoter, we
found that transfecting SMADs 3 and 4 into fibroblasts enhanced
CTGF promoter activity whereas SMAD7, but not SMAD6,
suppressed TGF
-induced CTGF expression. In SMAD3-knockout fibroblasts (45), CTGF induction did not occur indicating that SMAD2
could not substitute for SMAD3. We also found that ski and snoN
oncoproteins, which bind SMADs thereby blocking
SMAD-dependent gene expression (46-48), attenuated the
TGF
-mediated induction of CTGF. That SMADs are involved with
induction of CTGF is not surprising because SMADs have been
specifically implicated in the TGF
up-regulation of matrix genes
(37, 51).
-mediated induction of CTGF in normal fibroblasts, we then
examined the role of SMAD-dependent TGF
signaling on the
constitutive CTGF expression observed in fibroblasts cultured from the
lesional area of scleroderma patients (12, 20). Previously, we have
shown that CTGF promoter activity is substantially elevated
in scleroderma fibroblasts relative to their normal counterparts (12).
Here, we found that the elevated level of CTGF promoter
activity observed in scleroderma fibroblasts is not dependent on the
SMAD binding site; mutating the SMAD binding site in the context of the
CTGF promoter does not decrease CTGF promoter
activity in scleroderma fibroblasts. Conversely the previously termed
TGF
RE (25) is required for basal CTGF promoter activity in normal fibroblasts and also for the elevated activity in scleroderma fibroblasts. The factor(s) binding this element are currently under
investigation, but are not AP-1, CREB, or Sp1; factors known to
contribute to TGF
responses in other contexts, because
oligonucleotides containing consensus binding sites for these factors
do not compete for protein binding to the TGF
RE in gel shift
studies.2 In any event, the
difference in gene expression patterns between normal and scleroderma
fibroblasts is not solely caused by activated SMAD-dependent TGF
signaling because the SMAD-responsive
PAI promoter (49, 50) is not elevated in scleroderma fibroblasts.
signaling. These data do not exclude the possibility that a cryptic SMAD site in the
CTGF promoter is used in the scleroderma fibroblasts;
however, sequence inspection of the CTGF promoter does not
yield another SMAD consensus binding site. In addition, cotransfection
of SMAD3 and 4 into dermal fibroblasts does not activate expression of
our SMAD mutant CTGF promoter/reporter construct (not
shown). Furthermore, TNF
treatment, which suppresses the
TGF
-induction of collagen and CTGF in a manner possibly involving
elevation of SMAD7 (20, 53-55), has no effect on basal collagen or
CTGF gene expression in scleroderma fibroblasts (20).
in acquisition and maintenance of the scleroderma
phenotype. For example, recent reports localized TGF
mRNA to the
leading edge of the scleroderma lesion, that is the region of enhanced
inflammatory response presumably involved with the initiation of the
fibrotic response, but not in the lesional area itself (9, 56, 57).
This is in contrast to CTGF whose expression patterns and levels are
highly correlated with the severity of fibrosis (44, 58). Similarly,
other studies that examined normal and scleroderma fibroblasts failed
to see elevated TGF
levels, enhanced binding of TGF
to cells, or
enhanced responsiveness to TGF
in scleroderma fibroblasts (10, 11,
12, 52). Combined with our data in this report, these results suggest
that although the initial expansion of the sclerotic lesion may require
TGF
action, the maintenance of the sclerotic lesion does not result from TGF
action per se. Rather, the maintenance of the
scleroderma phenotype may result from the failure to suppress
downstream responses to TGF
, such as the induction of CTGF and
collagen synthesis. Thus although anti-TGF
/anti-SMAD strategies may
be effective in blocking the onset or progression of fibrosis (see Ref.
6), these strategies may not be effective at reversing fibrosis once it
has occurred. Given the known profibrotic activity of CTGF, the high
level of constitutive, SMAD-independent, CTGF expression present in the
sclerotic lesion may be a cause of the excessive scarring that is
observed in scleroderma patients. Thus, an anti-CTGF strategy, or at
least identifying why CTGF is overexpressed in fibrosis, might yield a
more effective method of reversing the fibrotic process.
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ACKNOWLEDGEMENTS |
---|
We thank George Martin for critical review of the manuscript, Alain Mauviel and John Varga for technical advice, Joan Massague for SMADs 2, 3, 4, 6, and 7 expression vectors, Anita Roberts for SMAD3-knockout fibroblasts, Robert Weinberg for ski and snoN expression vectors, and Dan Rifkin for the PAI-1/luciferase construct.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant AR45879, Arthritis Research Campaign (UK), The Raynaud's and Scleroderma Association Trust, and the Nightingale Charitable Trust.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.
§ These authors contributed equally to this work.
To whom correspondence should be addressed: FibroGen, Inc.,
225 Gateway Blvd., S. San Francisco, CA 94080. Tel.: 650-866-7336; Fax: 650-866-7207; E-mail:aleask@fibrogen.com.
Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M010149200
2 A. Leask and S. Sa, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
TGF, transforming
growth factor-
;
TGF
RE, TGF
response element;
CTGF, connective
tissue growth factor;
DMEM, Dulbecco's modified Eagle's medium;
HA, hemagglutinin;
PAI-1, human plasminogen activator inhibitor;
SEAP, secreted enhanced alkaline phosphatase.
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