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Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts.

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ARTHRITIS & RHEUMATISM
Vol. 48, No. 7, July 2003, pp 1964–1978
DOI 10.1002/art.11157
© 2003, American College of Rheumatology
Expression and Regulation of Intracellular SMAD Signaling in
Scleroderma Skin Fibroblasts
Yasuji Mori, Shu-Jen Chen, and John Varga
Objective. Scleroderma is characterized by excessive synthesis and accumulation of matrix proteins in
lesional tissues. Transforming growth factor ␤ (TGF␤)
plays a central role in the pathogenesis of fibrosis by
inducing and sustaining activation of fibroblasts; however, the underlying mechanisms are poorly understood.
We undertook this study to examine the expression and
function of SMADs, recently characterized intracellular
effectors of TGF␤ signaling, in scleroderma fibroblasts.
Methods. Primary dermal fibroblasts obtained
from 14 patients with scleroderma and from 4 healthy
adult volunteers were studied. Northern analysis was
used to determine the expression of endogenous SMAD
messenger RNA (mRNA), and Western analysis was
used to determine SMAD protein expression. Intracellular compartmentalization of cellular SMAD proteins
in the presence and absence of TGF␤ was studied by
antibody-mediated immunofluorescence confocal microscopy. The effect of TGF␤ blockade on SMAD subcellular distribution was determined using anti-TGF␤
antibodies as well as a dominant-negative TGF␤ receptor type II (TGF␤RII) vector to disrupt TGF␤ responses. SMAD-regulated luciferase reporter expression was examined to investigate the potential
functional significance of activation and nuclear accumulation of endogenous SMADs in scleroderma fibroblasts.
Results. Protein and mRNA levels of SMAD3, but
not of SMAD4 or SMAD7, were variably elevated in
scleroderma fibroblasts compared with those from
healthy controls. In sharp contrast to control fibroblasts, which displayed predominantly cytoplasmic localization of SMAD3/4 in the absence of exogenous
TGF␤, in scleroderma fibroblasts SMAD3 and SMAD4
consistently showed elevated nuclear localization. Furthermore, phosphorylated SMAD2/3 levels were elevated and nuclear localization of phosphorylated
SMAD2/3 was increased, suggesting activation of the
SMAD pathway in scleroderma fibroblasts. Blockade of
autocrine TGF␤ signaling with antibodies or by expression of dominant-negative TGF␤RII failed to normalize
SMAD subcellular distribution, suggesting that elevated
nuclear SMAD import was due to alterations downstream of the TGF␤ receptors. The activity of a SMADresponsive minimal promoter–reporter construct was
enhanced in transiently transfected scleroderma fibroblasts.
Conclusion. This study is the first to demonstrate
apparently ligand-independent constitutive activation of
the intracellular TGF␤/SMAD signaling axis in scleroderma fibroblasts. SMAD signaling may be a mechanism contributing to the characteristic phenotype of
scleroderma fibroblasts and playing a role in the pathogenesis of fibrosis.
Scleroderma is associated with fibrosis of affected tissues due to excessive synthesis and progressive
accumulation of connective tissue macromolecules (1).
Scleroderma fibroblasts display features of sustained
activation in vitro and in vivo. In culture, these cells show
elevated synthesis of collagens, fibronectin, proteoglycans, and tissue inhibitors of metalloproteinases, constitutive secretion of interleukin-1␣ (IL-1␣) and IL-6, and
reduced production of collagenase (for review, see ref.
2). These phenotypic alterations closely correspond to
changes in normal fibroblasts stimulated with transforming growth factor ␤ (TGF␤) and persist during serial
passage for a limited number of replications in vitro. The
mechanisms responsible for the activated phenotype of
scleroderma fibroblasts remain unknown.
Supported by NIH grant AR-42309 and by the Scleroderma
Foundation.
Yasuji Mori, MD, PhD, Shu-Jen Chen, PhD, John Varga,
MD: University of Illinois at Chicago College of Medicine.
Address correspondence and reprint requests to John Varga,
MD, Section of Rheumatology (M/C 733), University of Illinois
College of Medicine, Room 1158 Molecular Biology Research Building, 900 South Ashland Avenue, Chicago, IL 60607. E-mail:
jvarga@uic.edu.
Submitted for publication October 28, 2002; accepted in
revised form March 12, 2003.
1964
SMAD SIGNALING IN SCLERODERMA FIBROBLASTS
TGF␤, the principal mediator of physiologic tissue remodeling, is strongly implicated in the pathogenesis of scleroderma (3). The expression of TGF␤ is
elevated in scleroderma dermal fibroblasts, as well as in
monocyte/macrophages and other infiltrating inflammatory cells, even prior to the appearance of fibrosis
(4–11). Levels of TGF␤ receptor type I (TGF␤RI) and
TGF␤RII are also elevated on scleroderma fibroblasts
(12–14). Tissue expression of TGF␤ is prominent in
animal models of scleroderma, and blockade of TGF␤
signaling with neutralizing antibodies or naturally occurring TGF␤ antagonists is effective in reducing collagen
accumulation and ameliorating experimental fibrosis
(15,16). Furthermore, TSK mice that have been genetically engineered to be deficient for one TGF␤ allele
show marked reduction in the thickness of the dermis
and accumulation of matrix (17).
In fibroblasts, TGF␤ stimulates matrix production and induces its own synthesis (autoinduction) as
well as that of connective tissue growth factor (CTGF)
(18–20). Recent studies have shed light on the intracellular signaling mechanisms that mediate relevant profibrotic TGF␤ responses. Binding of TGF␤ to the ubiquitous type II serine/threonine kinase receptor
(TGF␤RII) triggers heterodimerization with and activation of TGF␤RI. The signal is then propagated downstream through SMADs, a family of evolutionarily conserved intracellular mediators that convey information
from the cell membrane into the nucleus (21). In
vertebrates, members of the SMAD family segregate
into 3 functionally distinct groups: SMADs 2 and 3 are
direct substrates for activated TGF␤RI, SMAD4 is the
common signaling partner, and SMAD7 is inhibitory.
Upon their phosphorylation and activation by the
TGF␤ receptor kinase at the cell membrane, SMAD2
and SMAD3 associate with SMAD4. The heteromeric
SMAD complex then translocates into the nucleus,
where, in conjunction with other DNA binding factors or
with transcriptional coactivators or corepressors, it regulates target gene transcription. While SMAD2 and
SMAD3 work synergistically and in tandem with
SMAD4 in transducing TGF␤ signals, the antagonistic
SMAD7 binds to the TGF␤ receptor complex and
prevents SMAD2/3 access, thereby blocking SMAD
phosphorylation. Because its expression is rapidly induced by TGF␤ (22–24), SMAD7 serves a critical negative feedback function by limiting the amplitude and
duration of cellular responses to TGF␤. In addition to
inhibitory SMAD7, multiple other intracellular mechanisms to modulate the intensity of SMAD signaling have
been characterized (25–28). In normal fibroblasts,
1965
SMAD3 and SMAD4 are predominantly cytosolic;
upon stimulation, these SMADs reversibly translocate
into the nucleus (24). Nuclear import is required for
SMAD-dependent transcriptional responses. The relative subcellular compartmentalization of SMAD3 and
SMAD4 is thus an important factor in setting the level of
TGF␤ signaling, but the mechanisms governing SMAD
nuclear–cytoplasmic shuttling in physiologic TGF␤ responses are poorly understood.
The SMAD pathway plays a fundamental role in
regulation of collagen synthesis, and SMADs are necessary to mediate TGF␤-dependent stimulation (29–33).
In light of the singular importance of TGF␤ in both
initiating and sustaining fibroblast activation, and given
the pivotal role of SMADs as major intracellular effectors of TGF␤-induced profibrotic responses, we undertook extensive characterization of SMAD signaling in a
large panel of scleroderma fibroblasts. The present
results indicate that messenger RNA (mRNA) and
protein expression of the positive signal mediator
SMAD3, but not those of inhibitory SMAD7, are variably elevated in scleroderma fibroblasts. Treatment with
TGF␤ caused repression of SMAD3 expression and
stimulation of SMAD7 expression in both healthy control and scleroderma fibroblasts. In sharp contrast to
control fibroblasts, however, scleroderma fibroblasts
were characterized by substantial increase in endogenous SMAD2/3 phosphorylation and nuclear accumulation in the absence of TGF␤ stimulation. Nuclear redistribution did not appear to be driven by autocrine
TGF␤, because blockade of TGF␤ signaling failed to
diminish the proportion of scleroderma fibroblasts
showing nuclear SMAD localization. Collectively, these
results demonstrate ligand-independent constitutive activation of the SMAD signaling pathway downstream of
the TGF␤ receptors in scleroderma fibroblasts, suggesting a novel mechanism that may contribute to the
pathogenesis of fibrosis.
PATIENTS AND METHODS
Cell culture. Primary dermal fibroblasts were established from excisional skin biopsy samples using previously
described procedures (29). In these experiments, 14 patients
with scleroderma were studied. The diagnosis of scleroderma
was established according to the classification criteria of the
American College of Rheumatology (formerly, the American
Rheumatism Association) (34). The mean duration of disease
at the time of biopsy was 12 months, and 3 patients were
receiving disease-modifying therapy (methotrexate, prednisone, or D-penicillamine). Biopsy samples were obtained
from the leading edge of clinically apparent “lesional” skin.
1966
MORI ET AL
Table 1. Clinical characteristics of patients with scleroderma
Patient/age/sex
1/56/F
2/35/F
3/46/F
4/59/F
5/39/F
6/61/M
7/50/M
8/38/F
9/47/F
10/43/F
11/19/F
12/19/F
13/30/F
14/44/M
Race*
Disease
duration,
years†
Skin involved,
%‡
B
H
B
O
W
W
H
W
W
W
H
B
W
W
1
1
1
1
1
1
2
1
1
1
0.5
1
1
1
20
10
5
5
50
5
50
36
40
5
40
15
10
20
* B ⫽ black; H ⫽ Hispanic; O ⫽ Oriental; W ⫽ white.
† Interval from first non-Raynaud symptom of scleroderma to time of
skin biopsy.
‡ Extent determined by clinical examination and recorded utilizing a
body surface diagram.
The characteristics of the patients are shown in Table 1. As a
control, skin biopsy samples were obtained from 4 healthy
adult volunteers (1 man and 3 women, mean age 45 years).
Biopsies were performed with written informed consent, and
the protocol was approved by the Institutional Review Board
of the University of Illinois.
Culture media were from BioWhittaker (Walkersville,
MD); all other tissue culture reagents were from Gibco BRL
(Grand Island, NY). For these experiments, control and
scleroderma fibroblasts were grown simultaneously and studied between passages 4 and 8. Cells were grown at 37°C in a 5%
CO2 atmosphere in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal calf serum (FCS), 1%
vitamin solution, 100 units/ml penicillin/streptomycin, and 2
mM L-glutamine. When the fibroblasts reached confluence,
fresh medium with indicated concentrations of TGF␤1 (Amgen, Thousand Oaks, CA) was added. In some experiments,
cultures were incubated with panspecific neutralizing antibody
against TGF␤ (R&D Systems, Minneapolis, MN) or nonimmune rabbit IgG as control, alone or in combination with
recombinant latency-associated peptide (LAP), a naturally
occurring TGF␤ antagonist (R&D Systems), for 0.5 hours
prior to addition of fresh medium. In other experiments,
scleroderma or control fibroblasts were transduced with adenoviral vector expressing green fluorescent protein (GFP) as
control, or with dominant-negative kinase-deficient TGF␤RII
to inhibit signal transduction from all 3 mammalian TGF␤
isoforms (35). Transduction was carried out by incubation of
confluent fibroblasts for 48 hours at room temperature with
3 ⫻ 109 plaque-forming units/ml of virus. Efficient transduction was confirmed by high levels of GFP expression in all
fibroblasts.
Extraction and analysis of RNA. At the end of each
experiment, total RNA was isolated from fibroblasts with
TRIzol reagent (Gibco BRL). Relative levels of mRNA were
examined by Northern analysis using ␥32P-dCTP–labeled com-
plementary DNA (cDNA) probes, as described (29). Following
extensive washing of the nitrocellulose membranes, the
cDNA–mRNA hybrids were visualized by autoradiography on
X-AR5 film (Eastman Kodak, Rochester, NY) exposed for
24–72 hours with intensifying screens. The following human
cDNA probes were used for hybridization: a 1.4-kb restriction
fragment that included the entire coding region of human
SMAD3, a 1.6-kb restriction fragment that included the entire
coding region of human SMAD4, a 1.9-kb restriction fragment
that included the entire coding region of human SMAD7,
GAPDH, and 18S. Signal intensities were quantitated by
densitometry, and results were normalized to the levels of
GAPDH or 18S mRNA in each sample.
Western analysis of cellular SMADs. Immunoblot of
whole cell lysates was performed as previously described (29).
To prevent protein dephosphorylation, a Phosphatase Inhibitor Mix (Sigma, St. Louis, MO) was added. Nuclear extracts
were prepared as previously described (31). Protein concentrations in the cytosolic, nuclear, or whole cell fractions were
determined by Bradford assay (Bio-Rad, Hercules, CA). Equal
aliquots of whole cell lysates (15 ␮g/lane) or cytosolic or
nuclear extracts (20 ␮g/lane) were separated by reducing
electrophoresis in 10% sodium dodecyl sulfate (SDS)–
polyacrylamide gels. Proteins in the gels were transferred onto
Immobilon-P (polyvinylidene difluoride) membranes (Millipore, Bedford, MA). Following blocking with 5% nonfat dry
milk in Tris buffered saline–Tween at room temperature for 1
hour, membranes were incubated for 2 hours with antibodies
against SMAD3 (Zymed, South San Francisco, CA) or against
SMAD2/3, phospho-SMAD2/3, SMAD4, SMAD7, TGF␤RI,
histone H3, or actin (all from Santa Cruz Biotechnology, Santa
Cruz, CA), followed by incubation with horseradish peroxidase
(HRP)–conjugated secondary antibodies. Antibody specificities were confirmed using blocking peptides supplied by the
manufacturer.
After washing, immunoblots were developed with
chemiluminescence reagents (Pierce, Rockford, IL) according
to the manufacturer’s protocol. In order to detect phosphorylated TGF␤RI or SMAD2/3 in fibroblasts, anti-TGF␤RI or
anti-SMAD2/3 antibodies with protein G–Sepharose were
used for immunoprecipitation. The immunoprecipitates from
lysates of fibroblasts left untreated or treated either with
TGF␤ (500 pM) or with anti-TGF␤ antibody were eluted by
boiling, electrophoresed through 8% SDS–polyacrylamide
gels, and processed as described above. Following overnight
blocking, membranes were incubated with antiphosphoserine
antibody (Zymed). Signal intensities were quantitated by densitometry, and results were normalized to the intensities of IgG
or histone H3 bands in each sample.
Cellular immunofluorescence imaging. The expression
and intracellular localization of endogenous SMAD proteins
were studied by immunocytochemistry and fluorescence confocal microscopy. For this purpose, fibroblasts (10,000/well)
from healthy controls or patients with scleroderma were
seeded into 8-well Lab-Tek II chamber glass slides (Nalge
Nunc International, Naperville, IL). The next day, fresh media
with 0.1% FCS and indicated concentrations of TGF␤1 were
added for 2 hours. Cells were washed and fixed with 100%
methanol for labeling of SMADs. Cells were then incubated
with antibodies (10 ␮g/ml) against SMAD2/3 (from Santa Cruz
Biotechnology or Zymed; both yielding identical results),
SMAD SIGNALING IN SCLERODERMA FIBROBLASTS
1967
SMAD4 or phospho-SMAD2/3 (both from Santa Cruz Biotechnology), or TGF␤RII (Santa Cruz Biotechnology) for 1
hour and then washed, followed by incubation with HRPconjugated secondary antibodies and staining with fluorescein
isothiocyanate or rhodamine according to the manufacturer’s
protocol (Tyramide Signal Amplification; NEN Life Science
Products, Boston, MA). To stain the nuclei, chambers were
mounted with Vectashield plus 4⬘,6-diamidino-2-phenylindole
(Vector, Burlingame, CA). Nonimmunized IgG was used as
negative control.
Following stringent washing of the slides, the pattern
and subcellular distribution of fluorescence were evaluated by
immunofluorescence or laser scanning confocal microscopy.
Each experiment was repeated at least 3 times with consistent
results. Quantitative analysis was performed by scoring 100
individual fibroblasts from different microscopic fields as
showing a predominantly nuclear or predominantly cytoplasmic distribution of immunofluorescence. The observer was
blinded to the identity of each section.
Transient transfection. Subconfluent cultures of fibroblasts were transfected using Superfect reagent (Qiagen, Valencia, CA), as described previously (35). The plasmid pSBE4luc (from Dr. B. Vogelstein), containing 4 tandem repeats of
an 8-bp palindromic consensus SMAD binding element (SBE)
that is specifically recognized by SMAD2 or SMAD3 (36), was
used to measure SMAD-mediated transcriptional responses.
pGL3-Luc, the empty vector of pSBE4-luc and pRL-TK (both
from Promega, Madison, WI) were used as internal controls to
correct for variations in transfection efficiency between the
samples. Following their recovery overnight in DMEM with
10% FCS, fibroblasts were incubated in fresh media containing
0.1% FCS. After 48 hours, cells were harvested and lysates
were prepared using passive lysis buffer (Promega). Luciferase
activity in equal aliquots was determined by Dual-Luciferase
Reporter Assay System (Promega). All experiments were
performed in triplicate and repeated at least 3 times. Significance of differences between experimental groups was determined by Mann-Whitney U test. P values less than 0.05 were
considered significant.
RESULTS
Expression and regulation of endogenous SMAD
mRNA in scleroderma fibroblasts. We previously demonstrated that in normal skin fibroblasts, SMAD3 and
SMAD4 played fundamental roles in mediating TGF␤
responses, including stimulation of collagen synthesis
(29,30). To begin to elucidate their role in the pathogenesis of fibrosis, we examined the expression of endogenous SMADs in scleroderma fibroblasts. Total
RNA was isolated from low-passage confluent fibroblasts and examined by Northern analysis. The results
showed that control and scleroderma fibroblasts similarly expressed two specific SMAD3 mRNA transcripts
of 7.0 kb and 3.0 kb (Figure 1A). Levels of SMAD3
mRNA were moderately elevated in scleroderma fibroblasts (mean ⫾ SEM 167 ⫾ 51% of those in control
Figure 1. SMAD mRNA expression in fibroblasts. Fibroblasts derived from lesional skin of 14 patients with scleroderma (S) or 4
healthy controls (N) were cultured in parallel. At early confluence,
cells were harvested and total RNA was isolated. Levels of SMAD3,
SMAD7, 18S, and GAPDH mRNA were determined by Northern blot
analysis. A, Representative Northern blot. Asterisks indicate the
positions of 18S and 28S ribosomal RNA. B, SMAD3 and SMAD7
mRNA levels were quantitated by densitometric scanning of the
autoradiographs. Results in the upper panel, corrected for the levels of
GAPDH mRNA in each sample, are expressed in arbitrary units. Each
data point represents the mean of several independent experiments for
each individual scleroderma (solid bars) and control (open bars)
fibroblast line. Lower panel shows the ratio of SMAD3 mRNA
level:SMAD7 mRNA level calculated for each fibroblast line. Middle
and upper/lower lines represent the mean ⫾ SEM.
fibroblasts), although the difference did not reach significance; in 11 of 14 scleroderma fibroblast lines,
SMAD3 mRNA levels were ⬎1 SEM higher than the
mean in controls (Figure 1B).
1968
In contrast to SMAD3, the expression of SMAD4
mRNA (9.5 kb and 3.9 kb) and SMAD7 mRNA (4.6 kb)
showed no consistent differences between control and
scleroderma fibroblasts, although SMAD7 mRNA levels
in 5 scleroderma fibroblast lines were ⬎1 SEM lower
than the mean in controls (Figure 1B). The ratio of the
mRNA levels of positive (SMAD3) and negative
(SMAD7) mediators of SMAD signaling (SMAD3
mRNA:SMAD7 mRNA ratio) was increased in scleroderma fibroblasts (mean ⫾ SEM 160 ⫾ 46% of the ratio
in control fibroblasts), although the difference was not
statistically significant. These results were reproducible
in several independent experiments, and they were
consistent when individual fibroblast lines were examined at different passages (passages 4 and 7).
In contrast to catalytic intracellular second messenger systems, the SMADs are devoid of intrinsic
enzymatic activity, and therefore the SMAD pathway
has no potential for amplifying input signals. Consequently, TGF␤-induced cellular responses are sensitive
to small changes in SMAD steady-state levels (37). We
therefore examined the regulation of cellular SMAD
mRNA expression by TGF␤. The results showed that
treatment with TGF␤ (500 pM) caused a selective
reduction in SMAD3 mRNA expression in normal fibroblasts. After 48 hours of treatment, the SMAD3 mRNA
steady-state levels were decreased to 38 ⫾ 6% (mean ⫾
SEM) of those in untreated fibroblasts. Failure to suppress SMAD3 expression could potentially result in
uncontrolled TGF␤ signaling due to a relative increase
in SMAD3 levels and persistent availability of SMAD3
for activation by the TGF␤ receptor kinase. We therefore examined whether the regulation of SMAD3
mRNA expression by TGF␤ was altered in scleroderma
fibroblasts. As shown in a representative Northern blot
(Figure 2A), SMAD3 steady-state mRNA levels in
scleroderma fibroblasts were markedly reduced by
TGF␤ (to 34 ⫾ 9% of the levels in untreated fibroblasts). An essentially identical magnitude of TGF␤induced decrease in SMAD3 mRNA was observed in 4
individual scleroderma fibroblast lines examined.
Inhibitory SMAD7 competes with SMAD3 for
access to the activated TGF␤ receptor, thereby preventing SMAD3 phosphorylation and blocking TGF␤induced responses in fibroblasts (29). Negative feedback
inhibition of TGF␤/SMAD signaling mediated through
endogenous SMAD7 limits the magnitude and/or duration of cellular responses to TGF␤, and rapid induction
of SMAD7 is therefore essential for preventing excessive
TGF␤ stimulation. Defective induction of cellular
SMAD7 results in unopposed SMAD signaling, leading
MORI ET AL
Figure 2. Regulation of SMAD mRNA expression by transforming
growth factor ␤ (TGF␤). Fibroblast lines obtained from lesional skin of
scleroderma patients or from skin of healthy controls were cultured in
parallel. When fibroblasts reached confluence, TGF␤1 (500 pM) was
added to the cultures. After the indicated period of incubation, cultures
were harvested and relative mRNA levels were determined by Northern
analysis. A, SMAD3 and SMAD7 mRNA levels determined in fibroblasts
incubated (solid bars in lower panel) or not incubated (open bars in lower
panel) with TGF␤ for 24 hours (SMAD3) or 90 minutes (SMAD7). An
autoradiograph representative of multiple independent experiments is
shown in the upper panel. Levels of mRNA were quantitated by densitometric scanning of the autoradiographs (lower panel). Results represent
the mean and SEM from 4 pairs of scleroderma (S) and normal (N)
fibroblast lines corrected for the levels of GAPDH mRNA in each
sample. B, Kinetics of SMAD7 mRNA induction by TGF␤ in control (left
panel) and scleroderma (right panel) fibroblasts. Note the difference in
scales between the two panels. Results for 7 individual scleroderma
fibroblast lines are shown.
to exaggerated or sustained TGF␤ responses. We
showed previously that transient disruption of endogenous SMAD7 function using antisense oligonucleotides
SMAD SIGNALING IN SCLERODERMA FIBROBLASTS
resulted in increased TGF␤ stimulation of COL1A2
transcription in normal fibroblasts (38). We therefore
examined the possibility that defective SMAD7 induction could account for the activated phenotype of scleroderma fibroblasts.
As shown in Figure 2A, TGF␤ induced a significant early rise in SMAD7 mRNA levels. Quantitation of
results from several independent experiments showed
that in scleroderma and control fibroblast lines treated
with TGF␤ for 90 minutes, SMAD7 mRNA levels were
increased to 261 ⫾ 42% and 256 ⫾ 35% (mean ⫾ SEM),
respectively, of those in untreated fibroblasts. Next, in a
separate set of experiments, we carefully compared the
kinetics of SMAD7 induction in control (n ⫽ 4) and
scleroderma (n ⫽ 7) fibroblast lines in parallel. As
shown in Figure 2B, there was a similar time course of
SMAD7 induction, with maximal TGF␤ response at 120
minutes both in control and in scleroderma fibroblast
lines. Significantly, none of the examined scleroderma
fibroblast lines failed to respond to TGF␤ with a ⬎2-fold
increase in SMAD7 mRNA expression. Taken together,
these results clearly indicate that control and scleroderma fibroblasts are characterized by comparable sensitivity to TGF␤, with similar magnitude and kinetics of
delayed down-regulation of SMAD3 mRNA expression
and rapid up-regulation of SMAD7 mRNA expression.
SMAD protein expression and activation in
scleroderma fibroblasts. The cellular steady-state levels
of SMAD proteins are determined by the rates of
transcription of the corresponding genes, as well as by
posttranscriptional regulatory mechanisms. SMADs are
subject to intracellular proteolysis mediated through the
ubiquitin ligase proteosome pathways in both liganddependent and ligand-independent manners (28).
Therefore, it was important to examine the levels of
SMAD proteins in control and scleroderma fibroblasts.
To this end, whole cell lysates were prepared from
confluent control and scleroderma fibroblasts in parallel
and subjected to immunoblot analysis.
In Figure 3A, a Western blot from a representative experiment demonstrates bands at ⬃52 kd (the
expected size of SMAD3) and at ⬃45 kd (the expected
size of SMAD7). Equal loading and transfer of protein
in each lane of the Western blots was confirmed using
antiactin antibody. Densitometric analysis of the results
demonstrated that 5 of 11 separate scleroderma fibroblast lines consistently displayed elevated SMAD3 protein levels compared with control fibroblasts. Generally,
a good correlation between relative levels of SMAD3
protein and mRNA expression was found in individual
fibroblast lines. In contrast to SMAD3, the levels of
1969
Figure 3. SMAD protein expression in fibroblasts. Fibroblasts from
normal control or lesional scleroderma skin were cultured in parallel.
At early confluence, cells were harvested and identical amounts of
whole cell lysates were electrophoresed and subjected to immunoblotting. A, A representative Western blot. Antibodies against SMAD2/3,
SMAD7, and actin were used. Numbers at the bottom indicate control
(N) or scleroderma (S) fibroblast lines. B, Increased SMAD phosphorylation in scleroderma fibroblasts. Whole cell lysates were prepared
from confluent control (lanes 1–3 [N3] and 4 [N1]) or scleroderma
(lanes 5 [S3], 6 [S5], 7 [S11], and 8 [S12]) fibroblasts in parallel. In
lanes 2 and 3, fibroblasts were incubated with transforming growth
factor ␤ for 30 minutes and 60 minutes, respectively. Proteins were
immunoprecipitated with antibody against SMAD2/3. The complexes
were electrophoresed through sodium dodecyl sulfate–polyacrylamide
gel, followed by immunoblotting with antiphosphoserine antibody
(p-Serine). Whole cell lysates were analyzed by immunoblotting for
IgG. A representative blot is shown. Numbers at the bottom indicate
the ratio of phospho-SMAD2/3 to total SMAD2/3 in each lane,
determined by quantitating the signal intensities by densitometry and
normalizing results to IgG to correct for small variations in protein
loading and transfer.
SMAD4 and SMAD7 proteins were similar in control
and scleroderma fibroblasts (Figure 3A and results not
shown). TGF␤ treatment resulted in a marked decrease
of SMAD3 protein at 48 hours and in an elevation of
SMAD7 protein at 4 hours both in control and in
scleroderma fibroblasts, whereas SMAD4 expression
remained unchanged. These changes in protein closely
paralleled the TGF␤-induced changes in SMAD mRNA
levels shown in Figure 2.
Upon TGF␤ stimulation, cellular SMAD2 and
SMAD3 become rapidly phosphorylated by the activated TGF␤ receptor serine/threonine kinase. The phosphorylation state of endogenous SMAD2/3 thus serves
1970
as a marker for the activation of the TGF␤/SMAD
pathway. We therefore sought to determine the relative
phosphorylation state of receptor-activated SMADs in
scleroderma fibroblasts. For this purpose, unstimulated
quiescent control and scleroderma fibroblasts in parallel
were lysed, and equal aliquots were immunoprecipitated
with anti-SMAD2/3 antibody, followed by electrophoresis and immunoblotting with antiphosphoserine antibody.
As shown in a representative immunoblot in
Figure 3B, only low levels of SMAD2/3 phosphorylation
could be detected in untreated control fibroblasts. Treatment of the fibroblasts with TGF␤ rapidly induced
phosphorylation of SMAD2/3, as expected. The expression levels of total SMAD2/3 proteins were unaffected
by treatment with TGF␤ for 60 minutes, as indicated by
immunoblotting with the anti-SMAD2/3 antibody. In
each of the scleroderma fibroblast lines studied, phosphorylated SMAD2/3 could be clearly detected in the
absence of TGF␤ stimulation (Figure 3B). Quantitation
of the immunoblot results from several independent
experiments indicated that the ratio of phosphorylated
SMAD2/3 to total SMAD2/3 in lysates from scleroderma
fibroblasts (n ⫽ 4) was 1.62 ⫾ 0.47, compared with
0.19 ⫾ 0.05 in control fibroblasts (n ⫽ 2) (Figure 3B and
data not shown).
Nuclear accumulation of SMAD3 and SMAD4.
Because SMAD-mediated TGF␤ signal transduction is
critically dependent on translocation of SMAD3 and
SMAD4 from the cytoplasm into the nucleus, the activity of the SMAD signaling pathway can be regulated by
the spatial compartmentalization of endogenous
SMADs. In order to examine subcellular SMAD distribution in scleroderma, SMAD3/4 nuclear accumulation
was analyzed using antibody-mediated immunofluorescence with confocal microscopy.
As shown in Figure 4A, SMAD3 showed predominantly nuclear distribution in ⬍10% of control fibroblasts in the absence of exogenous TGF␤. Treatment
with TGF␤ resulted in a rapid increase in SMAD
nuclear accumulation, which was maximal by 120 minutes (Figure 4A). Interestingly, we found that TGF␤
concentrations substantially below the threshold required for induction of transcriptional responses (such
as stimulation of COL1A2 and plasminogen activator
inhibitor 1) were capable of inducing SMAD3/4 nuclear
translocation (e.g., a concentration of TGF␤ as low as 10
pM was sufficient to induce nuclear accumulation of
endogenous SMAD3/4 in ⬎60% of normal fibroblasts
[24]).
MORI ET AL
Figure 4. Increased nuclear SMAD accumulation in scleroderma
fibroblasts. Control or scleroderma fibroblasts in parallel were
incubated with transforming growth factor ␤1 (TGF␤1), fixed, and
stained with fluorescein isothiocyanate (FITC)–conjugated
SMAD3- or SMAD4-specific antibodies. A, Immunofluorescence
confocal microscopic images from a representative experiment.
Green color (FITC) indicates SMAD4; blue color (4⬘,6-diamidino2-phenylindole) indicates the nucleus. a, Untreated control fibroblasts; b, fibroblasts treated with TGF␤; c, untreated scleroderma
fibroblasts; d, scleroderma fibroblasts treated with TGF␤ (original
magnification ⫻ 100 in upper panels; ⫻ 630 in lower panels). B, The
proportion of scleroderma or control fibroblasts unambiguously
displaying a predominantly nuclear pattern of SMAD3 or SMAD4
immunofluorescence in the absence of added TGF␤ was quantitated by scoring 100 individual cells in each culture in a blinded
manner. Results shown represent the mean and SEM from 5
individual scleroderma (solid bars) and 3 individual control (open
bars) fibroblast lines. ⴱ ⫽ P ⬍ 0.05. C, Nuclear accumulation of
SMAD2/3. Nuclear extracts were isolated from confluent scleroderma fibroblasts (S5, S12) or untreated or TGF␤-treated control
fibroblasts (N1) and examined by immunoblot using antibodies to
SMAD2/3 and histone H3.
SMAD SIGNALING IN SCLERODERMA FIBROBLASTS
Figure 5. Increased nuclear phospho-SMAD accumulation in scleroderma fibroblasts. Control or scleroderma fibroblasts incubated with
or without antibody (Ab) to TGF␤ in parallel were fixed and stained
with FITC-conjugated phospho-SMAD2/3 antibodies. A, Immunofluorescence confocal microscopic images from a representative experiment. Green color indicates phospho-SMAD2/3; blue indicates nuclei.
a, Control fibroblasts; b, scleroderma fibroblasts; c, scleroderma
fibroblasts incubated with anti-TGF␤ antibody (original magnification ⫻ 100 in upper panels; ⫻ 400 in lower panels). B, The proportion
of fibroblasts displaying predominantly nuclear localization of
phospho-SMAD2/3 in the absence of added TGF␤ was quantitated by
scoring 100 individual cells in each culture in a blinded manner.
Results shown represent the mean and SEM from 3 independent
experiments with 4 individual scleroderma (solid bars) and 3 individual
control (open bars) fibroblast lines. ⴱ ⫽ P ⬍ 0.05. See Figure 4 for
other definitions.
In contrast to control fibroblasts, 30–40% of
unstimulated scleroderma fibroblasts showed predominantly nuclear localization of SMAD3 in the absence of
exogenous TGF␤. The intensity of nuclear SMAD3
1971
immunofluorescence showed some variability among
individual fibroblasts. Elevated nuclear accumulation of
SMAD3 persisted during serial in vitro passages of the
scleroderma fibroblasts. Results were essentially identical when subcellular distribution of SMAD4 was compared between control and scleroderma fibroblasts. Incubation of fibroblasts with irrelevant IgG as negative
control resulted in minimal fluorescence. Quantitation
of results from multiple experiments with several
fibroblast lines indicated that the proportion of fibroblasts displaying predominantly nuclear SMAD3 localization was 3.3-fold greater in scleroderma cultures
(n ⫽ 6) than in control cultures (n ⫽ 3); for SMAD4,
this proportion was 3.7-fold greater in scleroderma
cultures (Figure 4B).
To confirm enhanced nuclear accumulation of
SMAD3 in scleroderma fibroblasts, nuclear extracts
were prepared from confluent cultures of control or
scleroderma fibroblasts in parallel and examined by
Western analysis. As shown in a representative immunoblot (Figure 4C), the accumulation of SMAD2/3 in the
nucleus was ⬃7-fold greater in scleroderma fibroblasts
than in control fibroblasts. As expected, treatment of
control fibroblasts with TGF␤ rapidly induced an ⬃10fold increase in nuclear SMAD accumulation. We next
compared the nuclear accumulation of phosphoSMAD2/3 in control and scleroderma fibroblasts. In the
absence of TGF␤, only relatively low levels of phosphoSMAD2/3 could be detected in control fibroblasts,
largely localized in the cytoplasm. In contrast, ⬃30% of
examined scleroderma fibroblasts showed strong expression and dominant nuclear localization of phosphoSMAD2/3 (Figures 5A and B).
SMAD7 acts as a component in negative feedback regulation of the fibroblast SMAD signaling pathway. By stably binding to the activated TGF␤ receptor,
SMAD7 competes with SMAD2/3 for receptor interaction (23). In previous studies, SMAD7 was shown to be
located predominantly in the nucleus and to translocate
into the cytoplasm upon stimulation by TGF␤. In those
experiments, transformed cell lines and recombinant
SMAD7 protein were used. We examined the subcellular localization of native SMAD7 in normal fibroblasts
by confocal microscopy. The results showed that in
contrast to transformed cells such as COS7, in normal
fibroblasts SMAD7 was located predominantly in the
cytoplasm, both in the absence and in the presence of
exogenous TGF␤ (results not shown). Therefore,
SMAD7 subcellular localization appeared to be cell type
dependent. The level of SMAD7 expression visualized
1972
by immunocytochemistry and its ligand-independent cytoplasmic localization were similar in control and scleroderma fibroblasts.
Effect of TGF␤ blockade on SMAD subcellular
distribution. Because TGF␤ is capable of inducing its
own production in fibroblasts, autocrine signaling may
contribute to sustaining of cellular responses to TGF␤.
Autocrine stimulation by endogenous TGF␤ has been
implicated in the constitutively activated phenotype of
scleroderma fibroblasts (12,13). Because autocrine
TGF␤ stimulation could account for SMAD activation
and nuclear accumulation observed in scleroderma fibroblasts, we examined the effect of blocking TGF␤mediated signaling by two complementary approaches.
For this purpose, first a neutralizing antibody recognizing all 3 isotypes of TGF␤ was used, either alone or in
combination with the naturally occurring TGF␤ antagonist LAP. Confluent fibroblasts were washed extensively to remove secreted TGF␤, fresh media containing
indicated concentrations of anti-TGF␤ antibody (1–20
␮g/ml) or nonimmune IgG were added, and the subcellular distribution of SMAD3/4 was then determined by
confocal microscopy.
The results showed that exposure of scleroderma
fibroblasts to the antibody for up to 48 hours, alone or in
combination with the TGF␤ antagonist LAP (5 ␮g/ml),
failed to down-regulate the level of nuclear SMAD4 or
SMAD3 (Figure 6A and data not shown). As an important control, ligand-induced SMAD nuclear import was
examined. As expected, addition of anti-TGF␤ antibody
resulted in a dose-dependent inhibition of SMAD4
nuclear translocation induced by TGF␤ in control fibroblasts. Anti-TGF␤ antibody (10 ␮g/ml) was capable of
reducing by ⬎70% the SMAD nuclear translocation
induced by TGF␤ at concentrations of up to 100 pM
(Figure 6A and data not shown). Therefore, failure to
reverse enhanced SMAD nuclear accumulation in
scleroderma fibroblasts by partial blockade of the autocrine TGF␤ activation loop suggests that a mechanism
independent of ligand-mediated TGF␤ receptor stimulation may have been responsible.
The effect of anti-TGF␤ antibodies on subcellular distribution of phospho-SMAD2/3 was characterized.
As shown in Figures 5A and B, ⬃30% of scleroderma
fibroblasts and ⬍5% of control fibroblasts showed a
predominantly nuclear localization of phosphoSMAD2/3, and anti-TGF␤ antibody failed to significantly reduce the proportion of scleroderma fibroblasts
showing nuclear phospho-SMAD2/3. Immunoblots of
nuclear extracts demonstrated elevated levels of
phospho-SMAD2/3 in scleroderma fibroblasts and fur-
MORI ET AL
Figure 6. The effect of blockade of transforming growth factor ␤
(TGF␤) signaling on SMAD nuclear accumulation. Confluent cultures
of fibroblasts were incubated with neutralizing antibody (Ab) to TGF␤
(␣-TGF-␤; 1–20 ␮g/ml) or with nonimmune IgG, followed by addition
of TGF␤ (10 pM) for 90 minutes. A, Cells were stained with SMAD4specific antibodies and examined by confocal fluorescence microscopy.
The proportion of control (open bars) or scleroderma (solid bars)
fibroblasts displaying a predominantly nuclear pattern of immunofluorescence was quantitated by scoring 100 individual cells in each
culture in a blinded manner. Results shown represent the mean and
SEM of duplicate determinations from 3 independent experiments. B,
Serum-starved normal (N1) or scleroderma (S5, S12) fibroblasts were
incubated with anti-TGF␤ antibody (10 ␮g/ml). Cells were harvested
and whole cell lysates (200 ␮g) were immunoprecipitated with antibody to TGF␤ receptor type I, followed by immunoblotting with
antiphosphoserine antibody (p-Serine). Membranes were stripped and
reprobed with antibody to IgG to confirm equal protein loading in
each lane.
ther confirmed the failure of anti-TGF␤ antibody to
“normalize” phospho-SMAD2/3 distribution (results not
shown).
SMAD SIGNALING IN SCLERODERMA FIBROBLASTS
In order to confirm that neutralizing anti-TGF␤
antibody was capable of blocking autocrine TGF␤ signaling, we examined the phosphorylation state of endogenous TGF␤RI (which is primarily phosphorylated on
serine residues in its GS domain upon activation by the
ligand) on untreated control and scleroderma fibroblasts. For this purpose, TGF␤RI was immunoprecipitated from whole cell lysates, followed by electrophoresis and immunoblotting with antiphosphoserine
antibodies. The results showed that while TGF␤RI was
constitutively phosphorylated in both control and scleroderma fibroblasts due to autocrine signaling, scleroderma fibroblasts exhibited ⬎2-fold higher TGF␤RI
phosphorylation than did control fibroblasts (Figure
6B). The levels of TGF␤RI were similar in scleroderma
and control fibroblasts (data not shown). Importantly,
treatment with anti-TGF␤ antibody caused a substantial
reduction in TGF␤RI phosphorylation in scleroderma
fibroblasts, but not in control fibroblasts. These results
indicate that under the conditions employed in these
experiments, anti-TGF␤ antibody effectively disrupted
the autocrine TGF␤ activation loop in scleroderma
fibroblasts.
To further determine the potential contribution
of autocrine TGF␤ signaling in enhanced SMAD nuclear localization, we used a dominant-negative
TGF␤RII vector to disrupt TGF␤ responses. The
dominant-negative TGF␤ receptor is unable to phosphorylate the type I receptor in response to any of the 3
TGF␤ isoforms, thus interrupting intracellular TGF␤
signaling. Normal and scleroderma fibroblasts in parallel
were transduced with the dominant-negative TGF␤RII
or empty vector, followed 48 hours later by incubation
with TGF␤ for 90 minutes.
As shown in a representative experiment in Figure 7A, in normal fibroblasts transduced with dominantnegative TGF␤RII, TGF␤-stimulated SMAD nuclear
accumulation was markedly reduced (compare panels b
and d). As expected, scleroderma fibroblasts showed a
markedly higher level of basal nuclear SMAD accumulation in the absence of added TGF␤ (panel e), and this
was not reduced by the dominant-negative TGF␤RII
(panel f). A high level of dominant-negative TGF␤RII
expression in the same cells was confirmed by immunocytochemistry (lower panels). Quantitation of the results
from several independent experiments confirmed the
ability of adenovirus-mediated dominant-negative
TGF␤RII overexpression to reduce by ⬎80% the
TGF␤-induced nuclear migration of SMAD2/3 in normal fibroblasts, as well as its failure to “normalize”
1973
Figure 7. The effect of adenovirus-mediated expression of dominantnegative transforming growth factor ␤ receptor type II (TGF␤RII) on
SMAD nuclear accumulation in scleroderma fibroblasts. A, Confluent
cultures of normal (a–d) or scleroderma (e and f) fibroblasts were
transduced with empty vector (a, b, and e) or adenovirus expressing
dominant-negative TGF␤RII (c, d, and f), followed 48 hours later by
addition of 10 pM TGF␤ (b and d). Following 90 minutes of incubation, fibroblasts were stained with antibodies to SMAD2/3 (upper
panels) or TGF␤RII (lower panels) and examined by confocal fluorescence microscopy. B, The proportion of control (open bars) or
scleroderma (solid bars) fibroblasts displaying a predominantly nuclear
pattern of SMAD3 immunofluorescence was quantitated by scoring
100 individual cells in each culture in a blinded manner. Results shown
represent the mean and SEM of duplicate determinations from several
independent experiments with 3 separate scleroderma cell lines. C,
Control (a) or scleroderma (b and c) fibroblasts were transduced with
empty vector (a and b) or dominant-negative TGF␤RII (c) and were
stained 48 hours later with antibodies to SMAD2/3 or TGF␤RII.
Immunofluorescence confocal microscopic images from a representative experiment are shown. In c, note that immunostaining confirms
substantial expression of dominant-negative TGF␤RII (green) in
scleroderma fibroblasts showing a high level of nuclear SMAD2/3
accumulation (pink). dnT␤RII ⫽ dominant-negative TGF␤RII. (Original magnification ⫻ 400.)
1974
MORI ET AL
activity of pGL3-luc was similar in lesional and control
fibroblasts.
DISCUSSION
Figure 8. SMAD-regulated transcriptional activity in fibroblasts. Subconfluent scleroderma (S) and control (N) fibroblasts in parallel were
transiently transfected with pSBE4-luc and appropriate control plasmids, and luciferase (Luc) activities were determined after 48 hours of
incubation. Results, normalized by renilla luciferase activity to correct
for small variations in transfection efficiency between samples, represent the means of triplicate samples from 3 independent experiments.
Bars show the group mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.05.
SMAD subcellular distribution in scleroderma fibroblasts (Figure 7B). Essentially identical results were
noted with 3 separate scleroderma cell lines. Transduction with GFP vector had no effect on either TGF␤induced SMAD nuclear migration in control fibroblasts
or constitutive nuclear accumulation in scleroderma
fibroblasts. Note the elevated nuclear accumulation of
SMAD2/3 in scleroderma fibroblasts showing high levels
of cellular dominant-negative TGF␤RII expression 48
hours posttransduction in the representative images
shown in Figure 7C.
SMAD-mediated transcriptional activity in
scleroderma fibroblasts. In order to investigate the
potential functional significance of ligand-independent
activation and nuclear accumulation of endogenous
SMAD3 and SMAD4 in scleroderma fibroblasts, we
examined SMAD-regulated luciferase reporter expression. The SMAD-responsive minimal pSBE4-luc construct was transiently transfected in control and scleroderma fibroblasts in parallel, and luciferase activity was
determined after 48 hours. As shown in Figure 8,
SMAD-inducible promoter activity was elevated in untreated lesional fibroblasts compared with controls. The
The recent discovery and characterization of the
SMAD protein family has provided novel opportunities
for delineating the involvement of TGF␤ in fibrosis (39).
In normal fibroblasts, SMADs function as essential
cytoplasmic effectors for TGF␤ stimulation of collagen
synthesis, as well as that of CTGF (40) and of TGF␤
itself (41). Genetic targeting of the SMAD3 locus protected null mice from the development of radiationinduced fibrosis, indicating the fundamental physiologic
role of SMAD3 in processes of tissue repair and fibrosis
(42). Because the activated scleroderma fibroblast phenotype is thought to reflect enhanced stimulation by, or
intrinsic responsiveness to, TGF␤, in the present study
we examined the expression and regulation of the
SMAD signaling pathway in these cells.
The results indicated that in a majority of scleroderma fibroblasts, SMAD3 protein and mRNA expression were elevated in comparison with control fibroblasts. Elevated levels of SMAD3 have also been
observed in hepatic myofibroblasts (43) and in a murine
model of renal fibrosis (44). Elevated SMAD3 expression could result in enhanced formation and nuclear
import of transcriptionally active SMAD heterocomplexes, with increased transcription of TGF␤-regulated
target genes. However, in light of the relatively modest
and variable increase in SMAD3 mRNA levels in scleroderma versus control fibroblasts (⬃60%), we consider it
unlikely that elevated SMAD3 expression plays a major
role in altered TGF␤ signaling in scleroderma fibroblasts.
Steady-state levels of SMAD3 are determined in
part by the rate of transcription of the SMAD3 gene and
the intracellular degradation of SMAD3 protein. The
factors determining the rate of SMAD3 transcription are
currently unknown. Because we found in the present
experiments that the increase in SMAD3 protein levels
in scleroderma fibroblasts compared with control fibroblasts was of relatively greater magnitude than the
increase in mRNA levels, it is possible that SMAD3
protein degradation was impaired; this is currently under
investigation. Importantly, the expression of SMAD3
mRNA in both normal and scleroderma fibroblasts was
selectively repressed by TGF␤. Inhibition of SMAD3
mRNA expression was a delayed response, with maximal
effect after 48 hours of exposure. These results clearly
indicate that TGF␤ can regulate the steady-state levels
SMAD SIGNALING IN SCLERODERMA FIBROBLASTS
of its own second messengers by pretranslational modulation, in addition to the well-documented proteasomal
degradation pathways.
Repression of SMAD3 mRNA levels in response
to TGF␤ has been noted in epithelial and mesangial
cells as well as in lung fibroblasts (45–47). In light of the
dependence of TGF␤ signaling intensity on the steadystate levels of its intracellular second messengers, inhibition of SMAD3 expression may thus be a negative
feedback mechanism with autoregulatory function to
prevent continuous SMAD signaling in response to
TGF␤ stimulation. The present results indicate that
elevated SMAD3 steady-state levels seen in some scleroderma fibroblasts cannot be attributed to failure of
TGF␤ to repress SMAD3 gene expression in these cells.
In normal cells, TGF␤ directly stimulates transcription of inhibitory SMAD7. Thus, SMAD7 induction
serves as a critical intracellular “brake” on TGF␤induced responses. The physiologic importance of this
SMAD7-mediated autoinhibitory feedback loop is highlighted by recent observations with cultured cells in in
vivo animal models. For example, tumor necrosis factor
␣–induced abrogation of TGF␤-dependent profibrotic
responses was linked to the induction of endogenous
SMAD7 (48). On the other hand, defective SMAD7
induction is implicated in the exaggerated TGF␤ responsiveness characteristic of hepatic cells and myofibroblasts from chronically injured livers (49,50). While the
highest level of SMAD7 expression is normally found in
the kidneys (23), in spontaneous renal fibrosis in
TGF␤1-transgenic mice, and in renal fibrosis induced by
anti–Thy-1 antibody, excessive matrix accumulation occurs in the setting of reduced SMAD7, resulting in
exaggerated or sustained local TGF␤ response (51,52).
Furthermore, a recent study demonstrated that SMAD7
basal expression levels and TGF␤-dependent inducibility were both markedly impaired in scleroderma fibroblasts in vivo and in vitro (53). These observations are
intriguing, because SMAD7 deficiency could provide a
mechanistic explanation for elevated TGF␤ receptor
expression and relative resistance to apoptosis that have
been reported in scleroderma fibroblasts.
In the present studies, we failed to detect consistent intrinsic alterations in basal levels of SMAD7
expression or SMAD7 inducibility. Protein and mRNA
expression of SMAD7, its regulation by TGF␤, and its
subcellular localization in scleroderma fibroblasts were
all comparable with those in normal fibroblasts. Therefore, our results do not support the hypothesis that in
scleroderma, fibroblasts may be “sensitized” to the
profibrotic effects of TGF␤ because of inability to
1975
“apply the brakes” on TGF␤-induced cellular responses
due to impaired SMAD7 regulation. It remains possible,
however, that despite its apparently normal inducibility
by TGF␤ in vitro, SMAD7 in scleroderma fibroblasts
somehow fails to inhibit receptor-mediated activation of
SMAD2/3.
In the absence of ligand stimulation, receptoractivated SMADs and SMAD4 reside mostly in the
cytoplasm, and their accumulation within the nucleus is
a key event in the intracellular propagation of TGF␤
signaling. Due to the presence of an N-terminal nuclear
localization signal sequence, SMAD3 displays intrinsic
nuclear import activity (54). In contrast, SMAD4 requires association with activated SMAD3 in order to
accumulate in the nucleus (55). In normal fibroblasts,
only a low level of nuclear SMAD accumulation could be
detected in the absence of exogenous TGF␤. In contrast,
the present studies revealed a substantial degree of
endogenous SMAD phosphorylation and nuclear accumulation in scleroderma fibroblasts. These findings were
highly consistent in each of the scleroderma fibroblast
lines studied.
Similar ligand-independent SMAD activation has
been described in hepatic stellate cells derived from
fibrotic livers (56) and in dermal fibroblasts derived
from keloid lesions (57). Constitutive SMAD activation
is therefore likely to contribute to the profibrotic phenotype of scleroderma fibroblasts. One of the significant
profibrotic effects of TGF␤ is its ability to inhibit
production of the matrix-degrading enzyme collagenase
1. Repression of collagenase in normal fibroblasts by
TGF␤ is mediated through SMAD3 (58). Therefore, the
present results demonstrating signal-independent activation of SMAD3 in scleroderma fibroblasts may provide a
mechanistic explanation to account for the reduced
collagenase expression previously described in scleroderma (59). Recent reports indicate that optimal induction of COL1A2 transcription by TGF␤ involves an
interaction between activated SMAD3 and the ubiquitous DNA binding transcription factor Sp1 (32,33). In
this regard, it is of interest that Sp1 has been shown to be
constitutively phosphorylated and activated in scleroderma fibroblasts (60). These findings suggest that for
maximal transactivation of COL1A2 in scleroderma
fibroblasts, SMAD activation and Sp1 phosphorylation
may both be required.
The mechanisms responsible for constitutive
SMAD activation in scleroderma fibroblasts are currently unknown. Autocrine stimulation by endogenous
TGF␤ may provide a possible explanation. Indeed, a key
role for autocrine stimulation by TGF␤ in the activated
1976
phenotype of scleroderma fibroblasts has been suggested, and elevated collagen synthesis was reduced by
disrupting endogenous TGF␤ signaling (12,13). It has
been recently demonstrated that SMAD3/4 undergo
continuous nucleocytoplasmic shuttling, and their relative levels within the nucleus are directly dictated by the
level of TGF␤ receptor activity (61). However, in the
present studies, we found that neutralizing anti-TGF␤
antibody and LAP failed to normalize SMAD subcellular distribution in scleroderma fibroblasts despite a
decrease in TGF␤RI phosphorylation. Furthermore,
transduction of dominant-negative TGF␤ receptor that
prevented TGF␤-induced SMAD activation in normal
fibroblasts failed to reduce SMAD nuclear accumulation
in scleroderma fibroblasts, suggesting that constitutive
SMAD nuclear accumulation was not due to autocrine
signaling by endogenous TGF␤.
It is conceivable that in the present experiments,
neither neutralizing antibody nor transduction of
dominant-negative TGF␤RII was able to completely
abrogate fibroblast activation mediated through endogenous TGF␤. In that case, the “ligand-independent”
SMAD activation we observed in scleroderma fibroblasts may in fact reflect autocrine TGF␤ stimulation.
This possibility cannot be ruled out, given that scleroderma fibroblasts in some studies (13,14), although not
in others (62), were shown to display elevated expression
of TGF␤ receptors and may thus have been “sensitized”
to TGF␤ (13,14); SMAD nuclear migration in fibroblasts can be induced by very low concentrations of
TGF␤. Alternatively, it is possible that non-TGF␤ ligands trigger cellular SMAD activation and nuclear
migration. For example, a recent report indicated that
insulin-like growth factor binding protein 3 (IGFBP-3)
could by itself induce SMAD2/3 phosphorylation and
potentiate TGF␤-stimulated cellular responses (63).
The ability of IGFBPs to induce TGF␤-independent
SMAD phosphorylation/activation may be particularly
relevant in the pathogenesis of scleroderma, since
IGFBP gene expression is increased in scleroderma
fibroblasts (64).
In summary, the present results provide evidence
for consistent alterations in the activation states of
intracellular TGF␤/SMAD signaling components in the
absence of exogenous TGF␤ in scleroderma fibroblasts.
These alterations may contribute to sensitizing scleroderma fibroblasts to TGF␤ or other stimuli that utilize
the SMAD pathway, resulting in enhanced fibrotic responses elicited by extracellular signals in pathologic
fibrosis.
MORI ET AL
ACKNOWLEDGMENTS
We are grateful to Dr. R. Derynck (University of
California, San Francisco), Dr. P. ten Dijke (Ludwig Institute
for Cancer Research, Uppsala, Sweden), Dr. J. Massague
(Memorial Sloan-Kettering Cancer Center, New York, NY),
and Dr. B. Vogelstein (Johns Hopkins University, Baltimore,
MD) for providing us with reagents, and we thank members of
our laboratory staff for helpful discussions.
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