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INTRODUCTION |
Transforming growth factor
(TGF)1-
is the prototypic
member of the TGF-
superfamily and mediates a multiplicity of
biological effects on different cell types. TGF-
regulates cellular
proliferation, induces synthesis of extracellular matrix proteins such
as fibronectin and plasminogen activator inhibitor-1 (PAI-1), modulates
the immune response, and plays an important role in embryonic
development and cellular differentiation (1).
TGF-
evokes its biological effects by signaling through two
different receptor serine/threonine kinases, TGF-
receptor type (T
R)-I and T
R-II, that form a tetrameric complex after binding of
TGF-
to T
R-II. T
R-II activates T
R-I by phosphorylation of
serine residues in the GS box. The anchor protein SARA (Smad anchor for receptor activation) recruits the cytoplasmic signal transducers Smad2 and Smad3, classified as so-called receptor-activated Smads (R-Smads), to the T
R-I kinase domain, resulting in their phosphorylation on serine residues in the C-terminal SSXS
motif. Activated R-Smads heteroligomerize with the common partner
(CO)-Smad4, and these complexes are transported into the nucleus, where
they regulate gene expression. R-Smads and CO- Smads contain two
highly conserved domains, the Mad homology (MH) 1 domain and the MH2 domain, which are connected by a linker region. Whereas their MH1
domains can interact with the DNA, the MH2 domains are endowed with
transcriptional activation properties.
Down-regulation of TGF-
signaling is effected, in part, by a
feedback mechanism involving induction of expression of the inhibitory
Smads, Smad6 and Smad7, which then prevent R-Smad activation (2,
3).
Absence of Smad2 or Smad3 expression resulting from targeted deletion
of the respective Smad genes in mice has revealed different developmental roles for Smad2 and Smad3. Homozygous loss of function mutations of the Smad2 gene by targeted deletion of the MH1
or MH2 domain resulted in embryonic lethality due to failure to
establish an anterior-posterior axis, gastrulation, and mesoderm
formation (4, 5). These events are controlled by
Smad2-dependent signals from the visceral endoderm (6, 7).
Postgastrulation-rescued Smad2 mutant embryos survived up to embryonic
day 10.5 but showed several malformations such as cyclopia, cranial
abnormalities, and impaired left-right patterning as observed by
abnormal heart looping and embryo turning (7).
In contrast, mice harboring homozygous deletions of the
Smad3 gene are viable and survive for several months,
indicating that Smad3 is dispensable for embryonic development.
However, Smad3 knockout mice are smaller than wild-type littermates and
show forelimb malformations (8, 9). Mice lacking expression of Smad3
die from chronic inflammation of several organs as a consequence of
impaired immune function including defects in mucosal immunity, as
revealed by abscesses in tissues adjacent to mucosal membranes, and
expansion of activated T-cell populations. This can be attributed in
part to the lack of responsiveness of Smad3-deficient T cells to the
growth-inhibitory effects of TGF-
as well as to a defective chemotactic response of Smad3-deficient neutrophils to TGF-
(8, 9).
Smad3-deficient mice show accelerated wound healing compared with
wild-type littermates, which is a consequence of enhanced re-epithelialization by proliferating keratinocytes and reduced wound
infiltration as well as TGF-
production by monocytes (10). Homozygous Smad3 knockout mice generated by Zhu et al. (11) die from colon carcinomas between 4 and 6 months of age, a phenotype that was not observed in Smad3 null mice derived by Datto et
al. (8) or Yang et al. (9).
To investigate the relative importance of Smad2 and Smad3 in TGF-
1
signaling, we have established mouse embryo-derived fibroblasts lacking
expression of the Smad2 or Smad3 gene (7, 9). In contrast to analysis of the function of Smad2 and Smad3 by
overexpression studies, these loss of function cell systems provide a
more appropriate model to investigate the physiological roles and
relative importance of these R-Smads in TGF-
signaling and provide
insight into the consequence of impaired TGF-
R-Smad function in
relation to pathophysiology. Our data show that expression of Smad2 or
Smad3 in fibroblasts is important for TGF-
1-mediated growth
inhibition as well as for synthesis of PAI-1, whereas Smad2 and Smad3
contribute uniquely to TGF-
1-induced activation of several
luciferase reporter constructs. We further show that certain genes are
selectively dependent on only one of these two TGF-
receptor-activated Smads, such as, for example, the matrix
metalloproteinase MMP-2, which is critically dependent on Smad2 but not
Smad3 expression. Collectively, our results indicate nonredundant roles
for Smad2 and Smad3 in TGF-
1-mediated signaling and provide insight
into the targets of these specific signaling pathways in
vivo.
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EXPERIMENTAL PROCEDURES |
Generation of Mouse Embryo-derived Fibroblasts and Primary Dermal
Fibroblasts--
Mouse embryo-derived fibroblasts harboring the null
allele Smad2
ex2 in the homozygous
state were derived as described previously (7). Briefly, embryonic stem
(ES) cells that were wild-type (Smad2+/+,
S2WT), heterozygous (Smad2
ex2/+),
or homozygous
(Smad2
ex2/
ex2,
S2KO) for the Smad2 null allele were established from
F1 heterozygous Smad2
ex2 intercrosses. ES
cells were on a C57/BL6 × 129V mixed genetic background.
Tetraploid blastocysts from ROSA 26, B6C3H, or CAF1 females were
derived following standard protocols and injected with S2WT or S2KO
ES cells. Embryos from 10.5 gestation days were triturated in
0.25% trypsin/1 nM EDTA and genotyped as described previously. Cells from two embryos with identical genotypes were pooled
and cultured, and one S2WT and one S2KO cell line survived and were
propagated in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum, 100 units/ml penicillin, and 50 µg/ml streptomycin to establish S2WT and S2KO fibroblasts that were cultured
over multiple passages to obtain sufficient cells to perform the experiments.
Smad3 knockout mice were generated by targeted deletion of exon 8 in
the Smad3 gene by homologous recombination as described previously (9). Mice heterozygous for the targeted disruption were
intercrossed to produce homozygous offspring. Embryos were triturated
in 0.25% trypsin/1 nM EDTA and genotyped as described previously (9). Cells from several Smad3 wild-type
(Smad3+/+; S3WT) and knockout
(Smad3
ex8/
ex8;
S3KO) littermate embryos (17 gestation days) were pooled by genotype to generate fibroblasts, which were used for experiments at
passage 3 as primary mouse embryo fibroblasts (MEFs) or routinely cultured over multiple passages in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and
50 µg/ml streptomycin to establish fibroblasts. The Smad2 and Smad3
fibroblasts used for the experiments shown were used at passage 20-35.
Three different S3KO and S3WT fibroblast lines were tested in the
different experiments, giving similar results; representative data are
shown. The results obtained were independent of the passage number.
Primary dermal fibroblasts (DFs) from S3WT and S3KO newborn pups were
established as described by Glick et al. (12) and used at
passage 2.
Mouse NMuMG mammary gland epithelial cells were cultured in Dulbecco's
modified Eagle's medium with 10% fetal bovine serum, 100 units/ml
penicillin, and 50 µg/ml streptomycin.
Western Blot Analysis--
Plasmids encoding Flag-tagged
Smad2 (pflSmad2) and Flag-tagged Smad3 (pflSmad3) were kindly
provided by Dr. J. Wrana (Mount Sinai, Toronto, Canada) and Dr. J. Massagué (Memorial Sloan-Kettering Cancer Center, New York, NY),
respectively. The expression construct encoding truncated Smad3 was
created as described previously (9). Recombinant adenoviruses
expressing Smad3 or
-galactosidase were obtained from Dr. K. Miyazono (The Cancer Institute, Tokyo, Japan) and used at MOI 40 to
infect primary S3KO MEFs and S3KO DFs as described by Fujii et
al. (13). Flag-tagged Smad2, Flag-tagged Smad3, and truncated
Smad3 were overexpressed in COS-1 cells, and proteins were extracted in
radioimmune precipitation buffer (125 mM NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 1% Triton X-100). S3WT and S3KO fibroblasts, MEFs, DFs, and lung tissue from S3WT
or S3KO transgenic mice were lysed in radioimmune precipitation buffer
to obtain protein extracts. Protein concentrations were measured using
the Bio-Rad protein assay according to the protocol provided by the
manufacturer. Protein samples (100 µg/lane) were separated by
SDS-polyacrylamide gel electrophoresis followed by wet transfer to
Immobilon-P membranes (Millipore). Nonspecific binding of proteins to
the membranes was blocked in TBS-T (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20) containing 5% milk.
Western blot analysis was performed using the rabbit polyclonal Smad2
or Smad3 primary antibodies (Zymed Laboratories Inc.)
and horseradish peroxidase-conjugated donkey anti-rabbit Ig secondary
antibody (Amersham Pharmacia Biotech). To detect MMP-2 in
gelatin-Sepharose-purified conditioned medium from the fibroblasts
(described below), a polyclonal rabbit anti-human MMP-2 antibody,
kindly provided by Dr. W. G. Stetler-Stevenson, was used (14).
Detection was performed by enhanced chemiluminescence.
Proliferation Assays--
Cell proliferation was measured by
[3H]thymidine incorporation as described by Danielpour
et al. (15). Briefly, WT, S2KO, and S3KO fibroblasts (1 × 104 cells/well) were seeded into 24-well dishes in
Dulbecco's modified Eagle's medium/10% fetal bovine serum. After
attachment (2 h), TGF-
1 was added to the culture medium for an
additional 24 h at varying doses, as indicated. The cells
were pulsed with 0.5 µCi/well [3H]thymidine for
the last hour of incubation, and incorporated label was determined as
described previously (15).
Northern Blot Analysis and cDNA Probes--
Total RNA was
isolated from cells using the RNeasy column purification method
(Qiagen, Santa Clara, CA) following the manufacturer's protocol. For
Northern blot analysis, RNA (10 µg) was electrophoresed on 1%
agarose gels and transferred to Nytran-N nylon membrane (Schleicher & Schuell). Membranes were hybridized with the relevant 32P-labeled cDNA probes in Church buffer (16) or
QuickHyb solution (Stratagene) according to the manufacturer's
protocol and analyzed by phosphorimagery or by exposure to Kodak BioMax
MS films. Equal RNA loading was assessed by ethidium bromide staining
of 28S and 18S rRNA, or, alternatively, membranes were hybridized with
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
or 18S rRNA cDNA probe. Hybridizations were performed
using the following cDNA inserts: 1300-bp
NheI/XhoI fragment of murine
p15INK4B, 420-bp EcoRI fragment of murine
p21CIP1/WAF1, 650-bp NdeI/XhoI
fragment of mouse p27Kip1, 2200-bp
BamHI/HindIII fragment of rat c-fos,
1300-bp EcoRI/XhoI fragment of murine
Smad7, and 1000-bp HindIII/XbaI
fragment of rat TGF-
1.
Extracellular Matrix Protein Assays--
TGF-
1-induced
fibronectin and PAI-1 synthesis was assayed as described by Wrana
et al. (17). MMP-2 was co-purified with fibronectin using
gelatin-Sepharose beads in the fibronectin assay. MMP-2 zymography was
basically performed as described by Kleiner and Stetler-Stevenson (18),
using gelatin-Sepharose affinity-purified proteins from
fibroblast-conditioned medium. Briefly, protein samples were separated
under nonreducing conditions on an 8% SDS-polyacrylamide gel
containing 1 µg/ml gelatin. The gel was incubated for 1 h at
room temperature in 2.5% Triton X-100 on a rotary shaker, followed by
incubation for 18 h at 37 °C in enzyme buffer containing 50 mM Tris, pH7.5, 200 mM NaCl, 5 mM
CaCl2, and 0.02% Brij-35. The gel was washed in
H2O, stained with Gelcode Blue Stain Reagent (Pierce,
Rockford, IL) according to the manufacturer's protocol, and dried on
3MM paper.
Transcriptional Reporter Assays--
3TP-Lux reporter construct,
ARE-luciferase reporter construct, and forkhead activin signal
transducer (FAST)-1 construct and (SBE)4-luciferase
reporter construct were provided by Dr. J. Massagué (Memorial
Sloan-Kettering Cancer Center), Dr. M. Whitman (Harvard Medical School,
Boston, MA), and Dr. P. ten Dijke (Ludwig Institute for Cancer
Research, Uppsala, Sweden), respectively. For transcriptional reporter
assays, fibroblasts were seeded at a density of 105
cells/6-well dish. The next day, cells were transfected with the
different luciferase reporter constructs using FuGene6 transfection reagent (Roche Molecular Biochemicals) according to the manufacturer's protocol. For Smad reconstitution experiments, pf1Smad2 or pf1Smad3 was
co-expressed with the respective luciferase reporter constructs. Cells
were transfected for 30 h followed by stimulation with the indicated concentrations of TGF-
1 for 18 h. In all
transfections, the expression plasmid pSV-
-galactosidase served as
an internal control to correct for transfection efficiency.
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RESULTS |
Analysis of Smad Expression in S2KO and S3KO
Fibroblasts--
Fibroblasts deficient in Smad2 or
Smad3 gene expression were derived from mice in which the
respective Smad alleles are disrupted by homologous
recombination resulting in targeted deletion of exon 2 in
Smad2 (7) and exon 8 in Smad3 (9), respectively. Western blot analyses of cellular lysates derived from S2KO fibroblasts and S2KO ES cells using four different antibodies against
various peptide sequences in the N-terminal and C-terminal domains of Smad2 demonstrated that the Smad2 deletion in exon 2 resulted in a null allele (7, 19). Western blot analysis of lysates from S3WT and S3KO fibroblasts, primary MEFs, primary DFs, or lung
tissue showed that S3WT cells and S3WT lung tissue express full-length
Smad3, whereas neither full-length nor truncated Smad3 protein could be
detected in S3KO cells or S3KO lung tissue using a specific antibody
that was raised against the Smad3 middle linker region (Fig.
1A). The antibody did detect
truncated Smad3 that was overexpressed by transient transfection in
COS-1 cells.

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Fig. 1.
Targeted deletion of Smad3 results in a null
allele. A, Western blot detection of Smad3 in protein
extracts from S3WT and S3KO mouse embryo-derived fibroblasts, primary
MEFs, primary DFs, and lung tissue. Primary S3KO MEFs and S3KO DFs were
infected with adenoviral Smad3 or adenoviral -galactosidase
( -gal) at MOI 40. Smad2, Smad3, and truncated
Smad3 were overexpressed in COS-1 cells, and protein extracts were used
as controls. The Smad3-specific antibody was raised against the linker
region of Smad3. B, effect of truncated Smad3 on
Smad2/FAST-1-mediated ARE reporter induction. NMuMG cells were
transfected with the ARE-luciferase reporter and FAST-1 in combination
with the indicated Smad proteins ( , control; , 80 pM
TGF- 1). Representative results are shown as the average of
triplicate observations corrected for transfection efficiency as
measured by -galactosidase activity.
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Although we were unable to detect expression of truncated Smad3 protein
in tissues or fibroblasts derived from S3KO mice, we assessed whether
expression of a putative truncated Smad3 protein could interfere with
Smad2 signaling by using a Smad2-dependent transcriptional
reporter assay (20, 21). Using NMuMG murine mammary gland epithelial
cells, we observed that TGF-
1 efficiently induced the ARE-luciferase
reporter in the presence of co-expressed forkhead activin signal
transducer FAST-1 and that this induction was further enhanced after
co-expression of Smad2 (Fig. 1B). Whereas overexpression of
full-length Smad3 abrogated the TGF-
1-dependent activation of the ARE reporter mediated by Smad2/FAST-1, as reported previously (22, 23), expression of truncated Smad3 enhanced ARE
reporter activation, presumably by its ability to act as a dominant
negative inhibitor of endogenous Smad3 (9). These data suggest that
even if it were expressed, this truncated version of Smad3 would not
interfere with Smad2-mediated TGF-
1 signal transduction (Fig.
1B).
Loss of Basal Proliferation and TGF-
1 Growth-inhibitory Response
in Smad2- and Smad3-deficient Fibroblasts--
Although TGF-
can
stimulate proliferation in several fibroblast cell lines, it is known
to inhibit the growth of primary MEFs (8, 24). We examined the
incorporation of [3H]thymidine to examine the roles of
Smad2 and Smad3 in control of basal rates of proliferation and in
transducing growth control signals by TGF-
1 in the different fibroblasts.
Basal rates of [3H]thymidine incorporation were 2-fold
higher in S2WT compared with S2KO fibroblasts and 8-fold higher in S3WT compared with S3KO cells (Fig. 2).
Inspection of cell cultures during the experiments did not reveal
significant differences in cell attachment, cell death, or cell
densities before and after labeling. These results indicate that lack
of either Smad2 or, in particular, Smad3 is associated with decreased
cellular proliferation rates in regular growth medium.

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Fig. 2.
Characteristics of cell growth responses in
Smad2- and Smad3-deficient fibroblasts.
[3H]Thymidine incorporation was measured in cpm in
different fibroblast genotypes in the absence ( ) or presence of 100 pM TGF- 1 for 24 h ( ). Representative experiments
performed in triplicate are shown. Error bars, S.D. from the
mean in triplicate experiments.
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In low-density cultures (10,000 cells/24-well dish) of S2WT or S3WT
fibroblasts, TGF-
1 treatment for 24 h reduced
[3H]thymidine incorporation by 46% (p = 0.001) and 54% (p = 0.01) when compared with untreated
cells, respectively (Fig. 2). In contrast, TGF-
1 had only a modest
effect on [3H]thymidine incorporation in S2KO (12%
reduction) and S3KO fibroblasts (16% reduction) compared with
untreated cells, respectively. At a higher plating density (15,000 cells/24-well dish), [3H]thymidine incorporation was
inhibited in response to TGF-
1 by 36% in S3WT and 9% in S3KO
fibroblasts and stimulated by 13% in S2KO fibroblasts compared with
untreated cells, respectively (data not shown). Our observations
demonstrate that lack of either Smad2 or Smad3 markedly reduces the
sensitivity of fibroblasts to growth inhibition by TGF-
1. However,
it should be noted that the relative absence of a growth-inhibitory
response in these cells is associated with already substantially
reduced basal growth rates when compared with the relevant WT fibroblasts.
Defects in p15 and p21 Regulation in Smad-deficient
Fibroblasts--
Regulation of the cyclin-dependent kinase
inhibitors p15INK4B, p21CIP1/WAF1, and
p27Kip1 has been shown to mediate cell cycle control by
TGF-
, depending on cell type and context (25). Because both S2KO and
S3KO fibroblasts showed reduced basal growth rates and had lost
growth-inhibitory responses to TGF-
1 (see Fig. 2), we examined the
effect of lack of Smad2 or Smad3 on cyclin-dependent kinase
inhibitor expression at baseline and in response to TGF-
1.
Northern blot analysis showed that TGF-
1 treatment of S2WT
fibroblasts resulted in early repression (3.9-fold) of p15 mRNA at
1 h but a 2.1-fold induction at 10 h. A similar profile was observed in S3WT fibroblasts after TGF-
1 treatment, with a 1.8-fold induction at 24 h (Fig.
3A). Baseline p15 mRNA
levels were reduced by 33% in S2KO fibroblasts and reduced by 35% in
S3KO fibroblasts compared with their WT controls, respectively, and
treatment of fibroblasts with TGF-
1 stimulated p15 expression in the
absence of Smad2, but not in the absence of Smad3. Importantly,
mRNA levels in both S2KO and S3KO fibroblasts in response to
TGF-
1 remained significantly reduced when compared with WT cells at
all time points examined, in particular at 10 and 24 h.

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Fig. 3.
Expression of inhibitors of
cyclin-dependent kinases in response to
TGF- 1 in fibroblasts deficient in Smad2 or
Smad3. Northern blot analysis of (A)
p15INK4B, (B) p21Waf1/Cip1, and
(C) p27Kip1 steady-state mRNA expression in
total RNA lysates from S2WT, S2KO, S3WT, and S3KO fibroblasts exposed
to 100 pM TGF- 1 for the indicated time periods.
Membranes were probed for glyceraldehyde-3-phosphate dehydrogenase
mRNA levels to normalize for RNA loading. Bar graphs
show the relative steady-state expression levels of
cyclin-dependent kinase inhibitors measured by densitometry
after normalization for glyceraldehyde-3-phosphate dehydrogenase ( ,
S2WT; light gray bars, S2KO; dark gray bars,
S3WT; , S3KO). Representative exposures from at least two
independent repeats per experiment are shown.
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In striking contrast with p15 expression, baseline mRNA levels of
p21 were dramatically increased in S2KO (22-fold) and S3KO fibroblasts
(9.3-fold) compared with WT cells (Fig. 3B). In both S2WT
and S3WT fibroblasts, TGF-
1 gradually induced mRNA levels of
p21, peaking at 5.4-fold and 5.1-fold induction at 10 h when compared with baseline. In contrast, TGF-
1 had little effect on the
already elevated levels of expression of p21 mRNA in S2KO cells. In
S3KO fibroblasts, TGF-
1 induced a 2.7-fold increase in expression at
1 h but only a modest increase of 1.4-fold at 10 h compared
to baseline. However, absolute p21 mRNA levels were considerably
higher at all time points in both KO cell types compared with the
respective WT fibroblasts.
Neither baseline mRNA levels of p27 nor the response of p27
mRNA expression to TGF-
1 treatment was significantly altered in
S2KO or S3KO fibroblasts compared with their respective WT controls
(Fig. 3C). In all genotypes, TGF-
1 treatment reduced p27
mRNA levels. In addition, expression of c-myc and p53 mRNA was
not affected by differences in genotypes or TGF-
1 treatment (data
not shown).
Taken together, these observations indicate that Smad2 and Smad3 are
each necessary in mediating the effects of TGF-
1 on cell
proliferation in fibroblasts established from mouse embryos and that
the absence of either Smad2 or Smad3 severely disturbs both the basal
and the TGF-
-dependent regulation of
p15INK4B and p21CIP1/WAF1 expression.
Induction of the Immediate Early Genes c-fos and Smad7, as well as
Autoinduction of TGF-
1, Is Dependent on Smad3--
To ascertain
whether Smad2 or Smad3 is required for the induction of early response
genes by TGF-
1, we examined the expression profiles of
c-fos and Smad7. TGF-
1 strongly induced
steady-state mRNA levels of c-fos at 1 h in S2WT and S2KO
fibroblasts but had no significant effect on c-fos expression in S3KO
fibroblasts (Fig. 4A). Lack of
Smad2 had no significant effect on the induction of Smad7 by TGF-
1
compared with S2WT fibroblasts (Fig. 4B). TGF-
1-mediated increase of Smad7 mRNA was significantly reduced in S3KO
fibroblasts compared with S3WT fibroblasts at 1 h (2.7-fold
versus 4.9-fold, respectively) and at 4 h (1.1-fold
versus 2.9-fold, respectively). The importance of Smad3 in
induction of Smad7 mRNA expression by TGF-
1 was further
confirmed in primary MEFs and primary DFs. Thus, adenoviral-mediated
reintroduction of Smad3 expression in the S3KO cells (Fig.
1A) restored induction of Smad7 mRNA expression by
TGF-
1 (Fig. 4C), whereas infection with recombinant
adenovirus expressing
-galactosidase had no effect (data not shown).
These observations demonstrate that Smad3 plays an important role in the induction of c-fos and Smad7 expression by
TGF-
1.

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Fig. 4.
Expression of immediate early response
genes c-fos and Smad7 and
TGF- 1 autoinduction. Northern blot
analysis of (A) c-fos and (B) Smad7 steady-state
mRNA expression in total RNA lysates from S2WT, S2KO, S3WT, and
S3KO fibroblasts exposed to TGF- 1 for the indicated time periods
shown. Membranes were probed for glyceraldehyde-3-phosphate
dehydrogenase mRNA levels, and quantitative analysis of
normalized mRNA expression is shown in the bar graphs
( , S2WT; light gray bars, S2KO; dark gray
bars, S3WT; , S3KO). Representative exposures from at least two
independent repeats per experiment are shown. C, Smad7
steady-state mRNA expression in total RNA lysates from primary S3WT
and S3KO MEFs and DFs exposed to TGF- 1 for the indicated time
periods. Smad3 expression was reintroduced in S3KO cells by adenoviral
infection at MOI 40. Ethidium bromide-stained 18S rRNA bands are shown
as RNA loading controls. D, TGF- 1 steady-state mRNA
expression in total RNA lysates from S2WT, S2KO, S3WT, and S3KO
fibroblasts exposed to TGF- 1 for the indicated time periods.
Membranes were probed for 18S rRNA to control for RNA loading.
E, TGF- 1 steady-state mRNA expression in total RNA
lysates from primary S3WT and S3KO MEFs and DFs exposed to TGF- 1 for
the indicated time periods. Smad3 expression was reintroduced in S3KO
cells by adenoviral infection at MOI 40. Ethidium bromide-stained 18S
rRNA bands are shown to control for RNA loading.
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It is well known that TGF-
1 can induce its own gene expression, in
part through the Ras/mitogen-activated protein kinase signaling pathway
that impinges on the transcriptional activation complex activator
protein 1 (AP-1) (26, 27). Analysis of TGF-
1 autoinduction in the
fibroblasts revealed that both basal and autoinduced expression of
TGF-
1 was strongly suppressed in S3KO cells, in contrast to S2KO
cells (Fig. 4D). This observation was further confirmed in
primary S3WT and S3KO MEFs and DFs. Thus, adenoviral reintroduction of
Smad3, but not
-galactosidase (data not shown), in the primary S3KO
MEFs and DFs restored autoinduction of TGF-
1 to levels observed in
S3WT cells (Fig. 4E). Due to early embryonic lethality of
the S2KO mice and the technical complications in deriving embryonic
fibroblasts, we were unable to perform similar experiments for primary
S2WT and S2KO MEFs and DFs. In conclusion, these data show that Smad3
plays an important role in autoregulation of
TGF-
1 expression in both primary and
established fibroblast cell cultures.
TGF-
1-induced Expression of Extracellular Matrix Proteins Is
Partially Dependent on Smad2 and Smad3 Expression--
TGF-
is
known to induce synthesis of several extracellular matrix proteins,
including fibronectin and PAI-1, in many different cell types, and
these have recently been shown to be Smad-independent and
Smad-dependent, respectively (8, 28). As shown in Fig. 5A, induction of fibronectin
synthesis in WT fibroblasts by TGF-
1 was similar to that in S2KO or
S3KO fibroblasts, consistent with the observation that Smad4 is
dispensable for TGF-
1-induced fibronectin synthesis and that
expression is instead dependent on expression and activation of c-Jun
N-terminal kinase (28). However, the possibility that Smad2 and
Smad3 can substitute for each other in induction of fibronectin
synthesis cannot be ruled out.

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Fig. 5.
Effect of TGF- 1 on
extracellular matrix synthesis in S2WT, S2KO, S3WT, and S3KO
fibroblasts. A, TGF- 1-induced fibronectin synthesis
in the different fibroblasts. Cells were serum-starved in the presence
of the indicated concentrations TGF- 1 for 18 h, and then cells
were metabolically labeled for 4 h with
[35S]methionine in the presence of the indicated
concentrations TGF- 1. Conditioned medium was collected, and
fibronectin was affinity-purified using gelatin-Sepharose beads,
followed by separation on 6% SDS-polyacrylamide gels. A 72-kDa protein
corresponding to MMP-2 was co-purified. B, identification of
the 72-kDa TGF- 1-induced gelatin-Sepharose-binding protein as MMP-2.
MMP-2 protein was induced by TGF- 1 as assessed by zymography,
metabolic labeling, and MMP-2 Western blot analysis, using
gelatin-Sepharose-purified conditioned medium from fibroblasts as
described above. C, TGF- 1-induced PAI-1 production in
S2WT, S2KO, S3WT, and S3KO fibroblasts, as well as in primary S3WT and
S3KO MEFs and DFs. Smad3 expression was reintroduced in S3KO cells by
adenoviral infection at MOI 40, and adenoviral -galactosidase
( -gal) infection at MOI 40 was done in
parallel as a control. Cells were treated with the indicated
concentrations TGF- 1 for 5 h, and [35S]methionine
was added during the last 3 h. Extracellular matrix proteins were
extracted and separated on 8% SDS-polyacrylamide gels. Experiments
were performed at least three times, and representative results are
presented.
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As shown in Fig. 5A, a TGF-
1-induced 72-kDa protein
co-purified with fibronectin in these assays. Based on its molecular mass, the fact that it was secreted into the conditioned medium, its affinity for gelatin, and its inducibility by TGF-
1, we
hypothesized that this protein could possibly represent collagenase
IV/72-kDa gelatinase/MMP-2, which has been reported to be induced by
TGF-
in fibroblasts (29). Indeed, we could detect the 72-kDa protein using an MMP-2 antibody, whereas its gelatinase activity was shown by
zymography (Fig. 5B). Interestingly, TGF-
1-enhanced
expression of MMP-2 was Smad2-dependent and did not require
Smad3. In contrast, tissue inhibitor of metalloproteinase 2, which
associates with MMP-2 and can therefore also be isolated using
gelatin-Sepharose beads, is known to be induced by TGF-
in several
cell types, and its induction by TGF-
1 appeared to be dependent on
both Smad2 and Smad3 (data not shown).
TGF-
1-mediated PAI-1 protein synthesis was also reduced in both S2KO
and S3KO fibroblasts, compared with WT cells (Fig. 5C). The
importance of Smad3 in induction of PAI-1 protein expression by
TGF-
1 was further validated in primary MEFs and DFs, where adenoviral reintroduction of Smad3 expression in primary S3KO fibroblasts resulted in full recovery of PAI-1 induction by TGF-
1. As mentioned above, the early embryonic lethality of S2KO mice precluded us from performing similar experiments in primary S2KO fibroblasts. Thus, for the most part, Smad2 and Smad3 have overlapping roles in TGF-
1-mediated induction of extracellular matrix proteins.
Role of Smad2 and Smad3 in Activation of TGF-
-induced
Transcriptional Reporters--
Receptor-activated Smad proteins
including Smad2 and Smad3 function as transcriptional regulators in the
nucleus. Whereas overexpression studies have shown that both Smad2 and
Smad3 can activate transcription of a variety of
TGF-
-dependent luciferase gene reporters, only Smad3 can
directly interact with Smad binding elements found in the promoters of
many TGF-
-responsive genes (30-33). To investigate the effect of
loss of each of these two TGF-
-activated R-Smads on the
transcriptional regulation of TGF-
-sensitive reporter genes, we
performed transfection studies in the fibroblasts. As shown in Fig.
6A, lack of Smad2 expression
only slightly reduced activation of the 3TP-Lux reporter (6.2-fold
versus 8.7-fold induction in S2KO versus S2WT
fibroblasts, respectively), which is driven by part of the PAI-1
promoter and three tetradecanoyl phorbol acetate-responsive elements
(17). In contrast, absence of Smad3 expression more strongly impaired
3TP-Lux reporter activation by TGF-
1 (2.2-fold versus
5.1-fold induction in S3KO versus S3WT fibroblasts; Fig.
6B). Reconstitution of Smad2 or Smad3 expression in the
respective S2KO or S3KO fibroblast cell lines restored TGF-
1-mediated activation of the 3TP-Lux reporter to levels achieved in WT cells (Fig. 6, A and B).

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Fig. 6.
Activation of
TGF- -responsive reporters in S2WT, S2KO, S3WT,
and S3KO fibroblasts. The effect of TGF- 1 on activation of the
3TP-Lux reporter (A and B), the
(SBE)4-luciferase reporter (C and D),
and the ARE-luciferase reporter (E and F) was
studied in S2WT versus S2KO fibroblasts (A, C,
and E), and in S3WT versus S3KO fibroblasts
(B, D, and F). FAST-1 was co-expressed with the
ARE-luciferase reporter. Reconstitution of Smad2 (A) or
Smad3 (B and D) expression in the respective
Smad-deficient fibroblasts resulted in recovery of induction of the
3TP-Lux reporter and (SBE)4-luciferase reporter ( ,
control; , 20 pM TGF- 1; , 80 pM
TGF- 1). Experiments were performed at least three times, and
representative results are shown as the average of triplicate values
corrected for transfection efficiency as measured by -galactosidase
activity.
|
|
TGF-
1-induced activation of the (SBE)4-luciferase
reporter, driven by four repeats of the CAGACA sequence identified as
Smad binding element in the JunB promoter (34), was
dependent on expression of Smad3, but not on expression of Smad2 (Fig.
6, C and D), consistent with the inability of
Smad2 to bind DNA. Reconstitution of Smad3 by transient overexpression
resulted in efficient activation of the (SBE)4-luciferase
reporter in S3KO fibroblasts. Similar results were obtained in primary
MEFs and primary DFs, where adenoviral-based reconstitution of Smad3
expression in S3KO fibroblasts also restored the ability of TGF-
1 to
induce (SBE)4-luciferase reporter activation (data not shown).
As reported previously (22) and described above (see Fig.
1B), Smad2 and Smad3 differentially affect TGF-
-induced
activation of the ARE-luciferase reporter when overexpressed. To
address the effect of loss of each of these Smad proteins, S2KO and
S3KO fibroblasts were transfected with the ARE-luciferase reporter and
FAST-1. Although the fold induction of ARE-luciferase reporter activity
by TGF-
1 was comparable in WT and S2KO fibroblasts, we repeatedly
observed that the overall ARE-luciferase reporter levels were
suppressed in S2KO fibroblasts (Fig. 4E). On the other hand,
absence of Smad3 expression enhanced both absolute levels of activation
and the fold induction of the ARE-luciferase reporter by TGF-
1 (Fig.
4F), supporting a suppressive role for endogenous Smad3 in
activation of the ARE-luciferase reporter (22). Together, these
reporter activation studies clearly indicate differential roles of
Smad2 and Smad3 in the induction of specific TGF-
1 target genes.
 |
DISCUSSION |
We have investigated TGF-
signaling in established mouse
embryo-derived fibroblasts deficient in expression of Smad2
or Smad3 to assess the effect of loss of each of these key
signaling intermediates on induction of target gene expression by
TGF-
1. We have identified target genes with Smad2- or
Smad3-independent patterns of induction, those that are affected by the
loss of either R-Smad, and genes that are selectively dependent on one
or the other of these two R-Smad proteins. As examples, we have shown
that TGF-
1-induced fibronectin synthesis occurs in the absence of
Smad2 or Smad3 expression, whereas both Smads have roles in the
induction of PAI-1 protein and in the more complex end point of
TGF-
1-induced growth inhibition with associated regulation of
cyclin/cyclin-dependent kinase inhibitors
p15INK4B and p21CIP1/WAF1. We
also show for the first time that TGF-
1-mediated induction of
c-fos expression requires Smad3 and that induction of MMP-2 is selectively dependent on Smad2. Moreover, similar to that shown for
Smad3 null macrophages and keratinocytes (10), we show that autoinduction of TGF-
1 in fibroblasts is
strongly suppressed in the absence of Smad3. To test that results shown
previously in overexpression systems are truly dependent on Smad2 and
Smad3, we have also assessed the activation of several
TGF-
-sensitive reporter genes in these Smad-deficient fibroblasts.
Together, these experiments demonstrate that Smad2 and Smad3 have both
overlapping and distinct roles in TGF-
1 signaling, depending on the
target gene and cellular context.
Because of the early embryonic lethal phenotype of the Smad2 knockout
mice (4, 5) and the technical difficulties involved in derivation of
S2KO embryonic fibroblasts, we were forced to do most of our
comparisons between the role of Smad2 and Smad3 in TGF-
1 signaling
using spontaneously immortalized fibroblasts that were cultured over
multiple passages. To underscore the validity of our studies, we show
that expression of Smad3 is also important for TGF-
1-mediated
induction of Smad7, TGF-
1, and PAI-1 in primary MEFs and primary
DFs. We also show that induction of expression of these genes by
TGF-
1 can be restored after stable reintroduction of Smad3 in these
primary Smad3-deficient fibroblasts. In contrast, whereas adenoviral-
or retroviral-mediated restoration of Smad2 or Smad3 expression in
fibroblasts could restore TGF-
1-responsive reporter gene induction
dependent directly on Smads, this strategy was not sufficient to
restore induction by TGF-
1 of endogenous gene responses shown to be
dependent on Smad2 or Smad3 (data not shown). Similar observations of
the inability to rescue responses by stable introduction of Smads or
other signaling molecules into established cell systems have been
reported (35, 36). Our data suggest that restoration of Smad expression
is not sufficient to fully revert the established Smad knockout
fibroblasts to their WT counterparts, possibly because loss of Smad
expression in combination with multiple genetic alterations, inherently
associated with immortalization, irreversibly alters expression of
additional genes important in mediating signaling to more complex
endogenous targets of TGF-
.
In contrast to previous reports that address the role of different
Smads by overexpression in in vitro systems, we show that fibroblasts derived from mouse embryos lacking expression of Smad2 or
Smad3 provide a suitable loss of function model system to investigate the different effects of these two R-Smads in TGF-
signaling, as is
important for the understanding of their distinct roles in
vivo. For example, the different roles of Smad2 and Smad3 are evident in studies of embryogenesis, where targeted deletion of Smad2
or Smad3 results in either early embryonic lethality or viable
offspring, respectively (4-9, 11). In wound healing, decreased levels
of Smad2 or Smad3 have dramatically different effects (10). Differences
are also apparent in carcinogenesis, where Smad2 has been classified as
a tumor suppressor based on its mutation frequency in several types of
cancer (37, 38), but where evidence for a similar role of Smad3 is
lacking (39, 40). Moreover, because autoinduction of TGF-
1,
previously shown to involve Ras/mitogen-activated protein kinase/AP-1
signaling (26, 27), is also dependent on Smad3 (Fig. 4, D
and E) (10), retention of Smad3 might be selected for in
tumor cells because TGF-
1 secreted by tumor cells can promote
tumorigenesis by inducing metastasis, invasion, and angiogenesis
(reviewed in Ref. 41). It should be noted, however, that R-Smad
activity can be blocked by certain oncogenes including Evi-1, which, in
certain cells, could have effects similar to its loss by genetic
defects (42).
A number of physical differences between Smad2 and Smad3 have been
described that might underlie or contribute to their observed functional differences or, in other cases, to their interchangeability. Whereas the MH1 domain of Smad3 can interact directly with SBE sequences (CAGAGTCT) in the DNA, Smad2 contains an extra exon that
encodes 30 amino acids absent in the MH1 domain of Smad3 and prevents
its binding to DNA (31, 32). Consistent with this, we observed that
TGF-
1-induced activation of the (SBE)4-luciferase reporter, which consists of four concatemerized SBEs derived from the
mouse JunB promoter (34), occurred as efficiently in S2KO as
in WT fibroblasts, whereas in S3KO cells, TGF-
1-induced
(SBE)4-luciferase reporter activation was impaired. This is
in agreement with previous observations that Smad2, in contrast to
Smad3, could not be detected in Smad-complexes bound to a
JunB probe and that overexpression of Smad2 contributed only
weakly to TGF-
1-induced activation of the
(SBE)4-luciferase reporter, whereas overexpression of Smad3 potently enhanced reporter induction, even in the absence of TGF-
(34). In a similar manner, Smad3 is involved in activation of the
Smad7 gene promoter, whereas Smad2 does not have a
functional role in its induction by TGF-
(19, 43). Interestingly,
mechanisms exist to alter the DNA binding patterns of Smad2 and Smad3.
An alternative splice variant of Smad2 that lacks exon 3 does bind to
DNA (32, 44) and might possibly compensate for loss of Smad3 in
mediating activation of certain TGF-
-induced responses.
Differential in vivo activities of Smad2 and Smad3 on the
same target element are also supported by our studies, suggesting that
Smad2 and Smad3 might have distinct affinities for different transcription factors and thereby contribute differently to TGF-
signaling. Thus, overexpression of Smad3 inhibits activation of the
goosecoid or Mix2 (ARE) TGF-
target gene promoters that are dependent on FAST, Smad2, and Smad4 (20, 21). This inhibition has been
proposed to result from either competition between Smad3 and Smad4 for
binding to FAST (23) or competitive affinities of Smad3 and Smad4 for
SBE elements in the gene promoters (22). Our loss of function studies
further support the dependence of this promoter on Smad2 as well as its
negative regulation by endogenous Smad3. Thus, we show compromised
activation of the ARE-luciferase reporter in Smad2-deficient
fibroblasts, in contrast with enhanced activation of this reporter in
Smad3-deficient cells as well as in NMuMG cells in which a truncated
dominant-negative form of Smad3 is overexpressed, likely interfering
with the function of the endogenous protein (9).
In contrast to direct activation of immediate early genes and
TGF-
-sensitive luciferase reporter genes that are likely controlled by low signaling thresholds, regulation of cell growth requires continuous signaling to modulate the tightly balanced cell cycle apparatus that integrates multiple signals at several complex end
points. It is therefore more difficult to identify the genes that are
directly involved in abrogation of TGF-
-induced inhibition of
cellular proliferation. Similar to our findings in Smad2- or Smad3-deficient fibroblasts, it has been reported that the
growth-inhibitory effects of TGF-
are lost in Smad3-deficient MEFs
and astrocytes (8, 45). Whereas Datto et al. (8) did not
observe changes in the induction of p15INK4B or
p21CIP1/WAF1 in Smad3-deficient MEFs, basal
expression levels of p15 and p21 were dramatically decreased and
increased, respectively, in our S2KO and S3KO fibroblasts compared with
WT controls. In agreement with this, suppressed p15 levels and reduced
activation of p15 by TGF-
have been observed in Smad3-deficient
astrocytes (45). However, the correlation between dysregulated p15 and
p21 levels and the observed growth behavior of our KO fibroblasts is
unclear at present.
The results presented here demonstrating that activation of genes by
TGF-
1 is often dependent on one of the two TGF-
R-Smads, Smad2 or
Smad3, suggest that the observed differences in KO phenotypes of Smad2-
versus Smad3-deficient mice are not merely a consequence of
differential spatially and temporally controlled patterns of gene
expression of these two R-Smads during development but rather reflect
unique, nonoverlapping roles for Smad2 and Smad3 in control of
target gene expression, which allows for more versatility in cross-talk
with other signal transduction pathways. Detailed analysis of TGF-
target gene expression in Smad2 versus Smad3 KO cell systems
now has the potential to provide insights into their respective roles
in regulation of genes that are of critical importance for both normal
physiology and development as well as in disease pathogenesis, including carcinogenesis.