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INTRODUCTION |
Transforming growth factor-
(TGF
)1 is a multipotent
cytokine that elicits many biological functions including inhibition of
the growth of cells of epithelial, endothelial, and lymphoid origins,
production of extracellular matrix components, and regulation of
differentiation of many cell types (1). These activities are mediated
by the cell surface types I and II TGF
receptors, T
RI and
T
RII, which are receptor serine/threonine kinases (2-4). In the
absence of ligand, while the T
RI kinase is inactive, the T
RII
kinase is constitutively active and the receptor is autophosphorylated (2, 5). Binding of TGF
1 to T
RII results in the formation of a
heteromeric complex containing T
RI and T
RII, followed by transphosphorylation of T
RI by the T
RII kinase (6).
Phosphorylation of T
RI by T
RII is thought to activate the T
RI
kinase activity, allowing it to phosphorylate and activate downstream
Smad2 (7-10) and Smad3 proteins (11, 12).
The Smad family proteins are critical components of the TGF
signaling pathway. Depending on their mechanisms of action, the Smads
can be divided into three classes: pathway-restricted Smads, common-mediator Smads, and inhibitory Smads (13). All Smad proteins share considerable homology in their primary sequence and most contain
two highly conserved Mad homology domains: MH1 in the amino-terminal
half and MH2 in the carboxyl-terminal half separated by a diverse
proline-rich linker. Upon stimulation by TGF
1, the pathway
restricted Smads, Smad2, and Smad3, interact with the TGF
receptor
complex, and become phosphorylated on three serine residues located at
the carboxyl termini of the molecules (9, 10). Phosphorylated Smad2 and
Smad3 then form a heteromeric complex with the common mediator Smad4
(14-16), translocate into the nucleus, and activate transcription of
TGF
responsive genes. As the common mediator, Smad4 plays a central
role in downstream signal transduction by the TGF
receptor family
members. By forming heteromeric complexes with various pathway
restricted Smads, Smad4 participates in the activation of multiple
signaling pathways initiated by TGF
family members and may be
directly involved in transcriptional activation of many downstream
genes (14, 16). As such, it plays a key role in the control of cell
growth and differentiation. Indeed, mutations that inactivate Smad4
function have been detected in many types of human cancer, including
cancers of the pancreas, colon, breast, neck, and stomach (17-21).
Importantly, many of these Smad4 mutants are defective in
transcriptional activation.
The mechanisms by which Smad proteins activate transcription of
specific genes are not well understood. The COOH-terminal MH2 domains
of Smad1 have been shown to act as transcriptional activators when
fused to a heterologous DNA-binding domain (22). Smad2, Smad3, and
Smad4 have been shown to interact with sequence-specific DNA-binding
proteins such as FAST-1 and FAST-2 and participate in the regulation of
transcriptional activation of specific promoters (23-25). Therefore,
it has been proposed that a sequence-specific DNA-binding partner is
required to bring the MH2 domain of the Smads into close proximity of a
TGF
responsive promoter in order to activate transcription. In
agreement with this model, overexpression of the MH2 domains of Smad2,
Smad3, or Smad4 is sufficient to activate transcription of the p3TPlux
luciferase reporter construct in a breast cancer cell line (14, 15).
Smad3 may cooperate with general transcription factors such as Sp1
(26), AP1 (27), and CBP/p300 (28, 29, 50) to activate transcription.
Recently, the NH2-terminal MH1 domains of
Drosophila Mad and human Smad3 and Smad4 were shown to
possess sequence-specific DNA binding activity (30-33, 49).
Interestingly, although Smad2 shares 91% sequence identity with Smad3,
it does not bind DNA. Using a PCR-based selection procedure, an 8-bp
palindromic DNA sequence (GTCTAGAC) was selected from a random pool of
oligonucleotides as the optimal Smad-binding element (SBE) for both
Smad3 and Smad4 (33). The crystal structure of the MH1 domain of Smad3
bound to SBE determined a minimal Smad box (GTCT) required for binding to Smad3 (34), although a high affinity interaction may require additional sequences. These results suggest that direct DNA binding of
Smad3 and Smad4 may play a role in transcriptional activation by
TGF
. However, since the involvement of the MH1 domains of Smads in
TGF
-induced transcriptional activation has not been demonstrated,
the exact role of the DNA binding activity of Smads in transactivation
is not clear.
The plasminogen activator inhibitor type-1 (PAI-1) promoter is the best
characterized TGF
-inducible promoter. Activation of PAI-1 gene
expression occurs mostly at the level of transcription with fast
kinetics (<30 min) and can reach up to 68-fold over the basal level
(35, 36). Deletional analysis suggested that there are multiple TGF
responsive elements present in the PAI-1 promoter that, together,
mediate optimal activation by TGF
. Among these elements, a 94-bp
region between
740 and
647 upstream of the initiation site was
found to be the major TGF
-responsive element that can mediate up to
50-fold activation of PAI-1 transcription (35, 36). Several AP1-like
sequences within this region have been proposed to play an important
role in mediating TGF
responsiveness (35). However, it is not clear
how TGF
induces transcriptional activation through this element.
Recently, Dennler et al. (32) reported identification of a
CAGA sequence found in three different locations in the promoter of the
PAI-1 gene that, when concatemerized three times, can mediate direct
binding of the Smad3 and Smad4 MH1 domains. Only one of the CAGA
containing sequences is located within this 94-bp major TGF
responsive element. Song et al. (37) reported that a 12-bp
sequence which overlaps with the above described CAGA sequence can
mediate Smad binding. We decided to carry out a detailed analysis of
this major TGF
responsive region to determine how many binding sites
there are in this region of the PAI-1 promoter and whether the
Smad-binding sites can mediate transcriptional activation in response
to TGF
. We have identified two specific Smad-binding sites in this
region which mediate cooperative binding of Smad3 and Smad4 and are
required for TGF
-induced transcriptional activation of the PAI-1
promoter. These two sites mediate a much stronger binding to Smads than
the previously reported CAGA containing sequences. A point mutation
identified in human pancreatic cancer that inactivates Smad4 function
was found to destroy the DNA binding activity of Smad4. Finally, we
showed that oligomerization of the Smad proteins are not required for
DNA binding.
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EXPERIMENTAL PROCEDURES |
Constructs--
Flag-tagged human Smad2 and Smad4 cDNA in
pCMV5B as well as Smad4 in pGEX-4T were kindly provided by J. Wrana and
L. Attisano (9). Human Smad3 cDNA was a generous gift from R. Derynck (11). To express full-length Smad3 and Smad3 and Smad4 mutants
in Escherichia coli, the Smad3 and Smad4 mutants were PCR
amplified and cloned into pGEX4T-1. NH2- and COOH-terminal
Smad mutants included the following amino acid residues: Smad4N,
1-318; Smad4C, 319-551; Smad3N, 1-201; and Smad3C, 230-426.
Mutations were generated using a PCR based approach and verified by
sequencing. p3TPlux was kindly provided by J. Massague (38). To
generate 4 × wild-type PAI-1(
740/
647), a DNA fragment
containing sequences between
647 and
740 from the PAI-1 promoter
(39) was PCR amplified with a BamHI site added at the 5' end
and a BglII site added at the 3' end. The PCR product was
digested with BamHI and BglII and self-ligated in
the same orientation. A fragment containing four copies of the PAI-1
promoter sequence in the same orientation was then cloned into
pGL2-promoter vector (Promega) at the BglII site. PAI-1
promoter sequences containing various point mutations were generated by PCR, and multimerized similarly as described for the wild-type promoter constructs.
Cells and Antisera--
293T cells were kindly provided by W. Pears (40) and maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum. Hep3B, a human hepatoma cell
line (ATCC), was maintained in minimum essential media supplemented
with 10% fetal bovine serum. TGF
1 is a kind gift from R&D systems.
The monoclonal antibody against the Flag tag (M2) was purchased from
Kodak and Sigma. The antibody against Smad2/3 (E-20) was purchased from
Santa Cruz Biotechnology. A polyclonal antibody against a
NH2-terminal peptide sequence of Smad4 was kindly provided by X. Liu.
Purification of GST Fusion Proteins--
GST fusion proteins
were expressed in and purified from E. coli as
described (41). Thrombin cleavage was performed with proteins bound to
glutathione-Sepharose (41). After elution, glycerol was added to
15% and proteins were stored at
80C.
Immunoprecipitation and Western Blotting--
Flag-tagged and
HA-tagged Smad proteins were isolated by immunoprecipitation from
transfected 293T cells as described (42). Briefly, 48 h after
transfection, cells were lysed in lysis buffer (50 mM Hepes
pH 7.8, 500 mM NaCl, 5 mM EDTA, 1% Nonidet
P-40, 3 mM dithiothreitol, and 0.5 mM
phenylmethylsulfonyl fluoride) and Flag- or HA-tagged proteins isolated
by immunoprecipitation with anti-Flag M2 affinity gel (Sigma) or
anti-HA affinity beads. Proteins were eluted from the antibody column
with the elution solution containing 1 mg/ml Flag or HA peptide (Sigma)
and quantified by Western blotting.
Transfection and Luciferase Assay--
293T cells were
transiently transfected using the calcium phosphate co-precipitation
method (5). Hep3B cells were transfected using a LipofectAMINE protocol
(Life Technologies, Inc.). For luciferase assay, a total of 2 µg of
DNA (1 µg of luciferase reporter construct and 1 µg of Smad
proteins) was used for each transfection. 24 h after transfection,
Hep3B cells were starved in serum-free media for 8 h and
stimulated with 50 pM TGF
1 for 16 h as described (26).
Electrophoresis Mobility Shift Assays (EMSA)--
For EMSA
assays, various amounts of recombinant proteins were incubated with
2 × 104 cpm 32P-labeled probes for 15 min
at room temperature in binding buffer (25 mM Tris-Cl, pH
7.5, 80 mM NaCl, 35 mM KCl, 5 mM
MgCl2, 10% glycerol, 1 mM dithiothreitol, 10 µg/ml poly(dI-dC), 300 µg/ml bovine serum albumin, and 2% Nonidet
P-40). The protein-DNA complexes were resolved on a 5% nondenaturing
polyacrylamide gel in 0.5% TBE. Double-stranded oligonucleotide probes
used in the EMSA assay are: Probe A (PAI-1 promoter region,
688/
660): wild-type: 5'- GAGAGTCTGGACACGTGGGGAGTCAGCCG-3'; M1
5'-GAGAGTCTGGACACGTGGGCATTAAGCCG-3'; M2
5'-GAGACATTGGACACGTGGGGAGTCAGCCG-3'; M3
5'-GAGACATTGGACACGTGGGCATTAAGCCG-3'. Probe B (PAI-1 promoter region,
675/
647): wild-type 5'-CGTGGGGAGTCAGCCGTGTATCATCGGAG-3'; M4
5'-CGTGGGCATTAAGCCGTGTATCATCG-3'; M5
5'-CGTGGGGAGTCAGCCGAATATACTCGGAG-3'. Probe C (PAI-1 promoter region,
732/
698): wild-type 5'GCCAGACAAGGTTGTTGACACAAGAGAGCCCTCAG-3'. Probe
D (PAI-1 promoter region,
740/
714): wild-type
5'-CAACCTCAGCCAGACAAGGTTGTTGAC-3'; M6
5'-CAACCTCAGCTATGTAAGGTTGTTGAC-3'.
Single-stranded oligonucleotides were end labeled using T4
polynucleotide kinase, annealed, and gel-purified (43). The complete PAI-1
740/
647 region was digested from p3TPlux using
BamHI and end labeled using T4 polynucleotide kinase.
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RESULTS |
Smad3 and Smad4 Bind to Two Adjacent DNA Elements in the PAI-1
Promoter in a Cooperative Manner--
EMSA were used to determine
whether Smad3 and Smad4 directly bind to the major TGF
responsive
region between
740 and
647 upstream of the initiation site in the
PAI-1 promoter. Recombinant full-length Smad3 and Smad4 were purified
from E. coli and incubated with the 32P-labeled
94-bp DNA fragment (probe FL). As shown in Fig.
1, both GST-Smad4 and GST-Smad3 bound to
this DNA fragment and induced a shift in the mobility of the labeled
probe (lanes 1 and 2, Fig. 1). To further
pinpoint the region that mediates Smad binding, four overlapping
oligonucleotide probes that cover the entire 94-bp region were
synthesized and tested in the EMSA assay (Fig. 1). Among the four
probes, probe A mediated strongest binding to Smad4 (lane
3, Fig. 1), while the other three probes all bound weakly
(lanes 4-6, Fig. 1). Therefore, the sequences between
688 and
660 covered by probe A contain the major binding sites for Smad4.
Similar results were obtained for Smad3 (data not shown). The CAGA
containing sequence that was previously reported to mediate Smad3
binding is covered by probes C and D. The 12-bp region that was
reported by Song et al. (37) to bind Smad3 was also included in probes C and D. Neither probes displayed a significant
affinity for Smads in our experiment (Fig. 1).

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Fig. 1.
Recombinant Smad3 and Smad4 bind to the 94-bp
major TGF -responsive region of the PAI-1
promoter. Top panel, probes used in EMSA assays. The
full-length 94-bp PAI-1 promoter region was end-labeled and incubated
with 0.4 µg of GST-Smad4 (lane 1) and 2 µg of GST-Smad3
(lane 2) in an EMSA assay. Lanes 3-6, 0.4 µg
of recombinant Smad4 purified from E. coli was incubated
with four oligonucleotide probes (A-D). Probe A contained
the major Smad4-binding sites.
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A careful examination of the DNA sequences between
688 and
660
revealed two potentially important sequence motifs: one is similar to
the SBE identified previously as the optimal Smad-binding sequence (33)
with a single A to G change at position 5 (SBE, GTCTAGAC; PAI-1
684/
677, GTCTGGAC), but the minimal Smad box is intact
(34). Six base pairs 3' to the SBE-like element is an AP1-like motif
which contains a single base pair change from the consensus AP1 site
(AP1, TGA(g/c)TCA; PAI-1
670/
664, GGAGTCA) (Fig.
2A). This
AP1-like site has been implicated in mediating TGF
-induced transcriptional activation of the PAI-1 gene (35) and
the type I collagen gene (44). The last four nucleotides in this
AP1-like motif is also homologous to the Smad box (GTCT) (34) with 1-bp
mismatch (GTCA). To determine whether the two motifs are required for
interaction with Smad4, oligonucleotide probes containing point
mutations in either of the two elements were generated (Fig.
2A) and their ability to complex with Smad4 was tested by
EMSA (Fig. 2B). Mutation of either the SBE-like site or
AP1-like site significantly reduced binding by GST-Smad4 (lanes
2 and 3, Fig. 2B), while mutation of both
site I and site II completely abolished binding of Smad4 (lane
4, Fig. 2B). This suggests that there are two
Smad4-binding sites in probe A and that both sites are important for
interaction with Smad4. Between the two sites, site I is the
predominant one since mutation of this site (probe M2) resulted in a
greater decrease in Smad4 interaction (lane 3, Fig.
2B). Interaction of Smad4 with site II, although weaker, was
also specific (see below and Fig. 3). As
a control, GST alone did not bind to probe A (lane 10, Fig.
2B). Furthermore, cleavage of GST by thrombin resulted in a
Smad4 that bound to probe A in a manner indistinguishable from that of
the GST-fusion proteins (lanes 5-8, Fig. 2B),
confirming that the observed DNA binding activity was not due to the
presence of GST.


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Fig. 2.
Smad3 and Smad4 bind to two adjacent DNA
regions in the PAI-1 promoter in a cooperative manner.
A, nucleotide sequence of wild-type and mutant probe A. SBE
and AP1-like motifs are capitalized and
underlined. Point mutations are indicated by
lowercase, bold-typed letters. B, EMSA assay
using 32P-labeled wild-type and mutant probe A. 0.4 µg of
Smad4 and Smad4N domain (including the MH1 domain and proline-rich
linker region) purified from E. coli either as a GST fusion
protein or cleaved with thrombin were incubated with the indicated
probes, and the protein-DNA complexes resolved on a 5% polyacrylamide
gel as described under "Experimental Procedures." The position of
unbound DNA probe is indicated. The exposure time was 1 h for
lanes 11-14 and 4 h for the rest. Lanes
1-4, GST-Smad4 incubated with wild-type and mutant probe A;
lanes 5-8, full-length Smad4 produced by thrombin cleavage
of GST-Smad4 incubated with wild-type and mutant probe A; lane
9, GST-Smad4C incubated with wild-type probe A; lane
10, GST incubated with wild-type probe A; lanes 11-14,
GST-Smad4N incubated with wild-type and mutant probe A. C,
1.6 µg of GST-Smad3 (lanes 1-4), GST-Smad3N (lanes
5-8), and GST-Smad3C (lane 9) were incubated with
wild-type and mutant probe A as indicated, and analyzed in an EMSA
assay as described. The gel was exposed for approximately 10 h.
D, cooperative binding of Smad4 to the two Smad-binding
sites. Indicated amounts of Smad4 produced by thrombin cleavage of
GST-Smad4 were incubated with wild-type or mutant probe A and analyzed
in an EMSA assay. The intensity of the DNA-protein complex was
quantified. E, Western blotting analysis of Flag-tagged Smad
proteins. Flag-tagged Smad proteins were isolated by
immunoprecipitation from transfected 293T cells, eluted with Flag
peptide, and quantified by Western blotting analysis using the
antibodies indicated. The Flag-Smad3 or Flag-Smad4 were compared with a
known quantity of recombinant Smad3 or Smad4 as indicated. F,
left panel, 0.4 µg of Flag-tagged full-length and truncated
Smad4 proteins isolated from transfected 293T cells were incubated with
32P-labeled probe A, and the ability of Flag-tagged
wild-type and mutant Smad4 to bind to DNA was tested by EMSA
(lanes 1-6). Antibody supershift was performed using 1 µg
of anti-Flag (M2) antibody (lane 4). The gel was exposed for
1 h. Right panel, approximately 1 µg of Flag-tagged
full-length and truncated Smad3 proteins isolated from transfected 293T
cells were subjected to EMSA assay with 32P-labeled probe A
(lanes 7-11). The gel was exposed for 3 h. Lane
12, a longer exposure (12 h) of lane 9.
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Fig. 3.
The 94-bp region from the PAI-1 promoter
contains multiple Smad-binding sites. A, Smad3 and
Smad4 specifically bind to site II. Top panel, nucleotide
sequence of wild-type and mutant probe B. The AP1-like motifs are
capitalized and underlined. Point mutations are
indicated by lowercase, bold-typed letters. Bottom
panels, GST-Smad4 (0.4 µg), GST-Smad4N (0.4 µg), GST-Smad3
(1.6 µg), and GST-Smad3N (1.6 µg) were used to test binding to the
32P-labeled probes as indicated. B, binding of
Flag-Smad3 and Flag-Smad4 to wild-type and mutant PAI-1 promoter
740/ 647 region and to wild-type and mutant probe D. EMSA was
performed as described using 0.4 µg of Flag-Smad4 and 1 µg of
Flag-Smad3 isolated from transfected 293T cells. Probes used:
lanes 1 and 3, full-length PAI-1 promoter
740/ 647 region (FL); lanes 2 and
4, PAI-1 promoter 740/ 647 region containing point
mutations in sites I and II (M); lanes 5 and 8, Flag-Smad4 and Flag-Smad3 binding to probe A; lanes 6 and
9, Flag-Smad4 and Flag-Smad3 binding to wild-type probe D;
and lanes 7 and 10, Flag-Smad4 and Flag-Smad3
binding to mutant probe D.
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The amount of Smad4 complexed with wild-type probe A was 4- or 5-fold
more than the amount complexed with probe M1 and M2 added together,
suggesting that the two binding sites act in a cooperative manner to
mediate binding of Smad4 (lanes 1-3 and 5-7,
Fig. 2B). A careful titration experiment using various
amounts of Smad4 in EMSA further confirmed the cooperative action
between the two sites (Fig. 2D). Mutation of site I or site
II also resulted in a decreased mobility shift, suggesting that Smad4
occupies both binding sites on the same DNA probe simultaneously. The
presence of multiple shifted bands in EMSA assay (lanes 2, 3, 6, and 7, Fig. 2B) could be due to the
different oligomeric states of Smad4 caused by GST fusion or
homoligomerization of Smad4.
Thus, recombinant Smad4 binds to two adjacent sites in the PAI-1
promoter in a cooperative manner. Similarly, the two sites also
mediated binding of GST-Smad3, albeit with a reduced affinity (lanes 1-4, Fig. 2C): at least 8 times more
GST-Smad3 was required to shift a similar amount of the DNA probe. The
cooperativity between the two sites in binding to Smad3 was also less
prominent than that observed in binding to Smad4 (Fig. 2, C
and E, and data not shown). Therefore, Smad3 and Smad4
displayed different binding properties for this region of the PAI-1 promoter.
Consistent with previously published studies, the N domains of Smad3
and Smad4 (GST-Smad3N and GST-Smad4N) were capable of binding to DNA
(lanes 11-14, Fig. 2B, and lanes
5-8, Fig. 2C). Neither GST-Smad3C nor GST-Smad4C bound
DNA (lane 9, Fig. 2, B and C).
To examine whether Smad3 and Smad4 isolated from mammalian cells also
interact with the PAI-1 promoter with a similar affinity, Flag-tagged
Smad3 and Smad4 were isolated by immunoprecipitation from transiently
transfected 293T cells, eluted with Flag peptides, and subjected to
EMSA analysis. The amount of Smad proteins prepared this way was
quantified by comparing to recombinant GST-Smad proteins using Western
blotting analysis (Fig. 2E).
As with bacterially expressed Smad3 and Smad4, Flag-Smad3 and
Flag-Smad4 isolated from transfected 293T cells bound to both site I
and site II in the PAI-1 promoter in a cooperative manner and with
affinities similar to those of the bacterially expressed proteins
(lanes 1-3, 7-9, and 12, Fig. 2F).
Addition of an anti-Flag antibody to the reaction induced a complete
supershift of the Smad4-DNA complex (lane 4, Fig.
2F), confirming that the observed DNA-protein complex
contained Flag-Smad4. Also similar to recombinant Smad proteins, the N
domains of Flag-Smad3 and Flag-Smad4 were responsible for DNA binding
(lanes 5 and 10, Fig. 2F), while the C
domains failed to bind (lanes 6 and 11, Fig.
2F). The N domain of Flag-Smad3 or GST-Smad3N bound probe A
with a higher affinity than the full-length protein (compare lane
7 with lane 10, Fig. 2F, and lane
1 with lane 5, Fig. 2C), suggesting that the
Smad3 C domain may inhibit DNA binding by the N domain. However, in contrast to Smad3, the N domain of Flag-Smad4 bound no better or even
weaker than the full-length Smad4 (compare lane 1 with lane 5, Fig. 2F). It is difficult to estimate the
amount of GST-Smad4N used in the EMSA assay due to the presence of many
shorter protein fragments co-purified with the full-length Smad4N
(lane 4, Fig. 2E). Since some of these fragments
may contain DNA binding activity, it is difficult to compare the DNA
binding activity between GST-Smad4N and GST-Smad4.
Taken together, our results suggest that Smad4 and to a lesser extent,
Smad3, purified either from bacteria or from mammalian cells, bind
specifically to two adjacent DNA elements in the PAI-1 promoter in a
cooperative manner, and this interaction is mediated by the
NH2-terminal domains of Smad3 and Smad4.
Smad3 and Smad4 Bind Specifically to Sequences Overlapping with the
AP1-like Site Located at
670/
664 in the PAI-1 Promoter--
Some
AP1 or AP1-like sites contain overlapping Smad boxes and have been
shown to mediate TGF
responsiveness (45). The TGF
-responsive region in the PAI-1 promoter between
670 and
653 contains two AP1-like sites (35). We have shown that efficient binding of Smad3 and
Smad4 to probe A requires sequences in the first AP1-like site. This
AP1-like site contains a degenerate Smad box (GTCA) with a single base
pair mismatch. The second AP1-like site located 3' to site II also
contains a degenerate Smad box (GTAT) (34). To determine whether Smad
proteins could recognize this sequence, a second set of probes (probe
B) that contain both AP1-like sites (Fig. 3A) were
synthesized and used in the EMSA assay. Both the full-length and N
domains of Smad3 and Smad4 bound to wild-type probe B as shown in Fig.
3A. Mutation of site II alone (M4) led to a complete loss of
binding (lanes 2, 5, 8, and 11, Fig.
3A), while mutation of the second AP1-like site (M5) did not
significantly affect Smad binding (lanes 3, 6, 9, and
12, Fig. 3A). This indicated that Smad proteins
did not bind to the second AP1-like site at
659/
653. Therefore,
Smads specifically recognize sequences in only one AP1-like site within
this 94-bp region of the PAI-1 promoter.
Additional Binding Sites within the 94-bp TGF
Responsive Region
of the PAI-1 Promoter--
To confirm that sites I and II are indeed
the major Smad-binding sites in the 94-bp region, a full-length probe
containing mutations in both site I and site II (probe M) was tested
for binding to Smad3 and Smad4 in the EMSA assay. Mutations of both site I and site II together greatly reduced binding by Smad4
(lane 2, Fig. 3B) and Smad3 (lane 4,
Fig. 3B), confirming that sites I and II are the predominant
Smad-binding sites, but that additional sequences outside the region
covered by probe A may also mediate a low affinity binding to Smads.
Since probes C and D showed a weak binding to Smads (Fig. 1), and the
region covered by both probes contains the CAGA sequence (
732 to
725) described by Dennler et al. (32) and the 12-bp
sequence by Song et al. (37) that interacts with GST-Smad3N
and GST-Smad4, this CAGA sequence is likely responsible for the
residual low affinity binding. Indeed, mutation of the CAGA site in
probe D completely abolished binding to Smad3 (lane 10, Fig.
3B) and Smad4 (lane 7, Fig. 3B).
Compared with probe A, binding of Flag-Smad3 and Flag-Smad4 to probe D was weaker by at least 10-fold (lanes 5, 6, 8, and
9, Fig. 3B). Thus, although the CAGA sequence can
mediate a low affinity binding to Smads, sites I and II are the major
sites recognized by Smad3 and Smad4. Taken together, these data
indicate that the 94-bp PAI-1 promoter region contains three
Smad-binding sites. This is consistent with a previous observation that
this region of the PAI-1 promoter contains multiple TGF
response
elements (35).
The Two Smad-binding Sites in the PAI-1 Promoter Are Essential for
TGF
-induced, Smad3-, and Smad4-dependent Transcriptional
Activation--
To determine whether the two Smad-binding sites we
identified are necessary for TGF
-induced activation of PAI-1 gene
expression, luciferase reporter constructs that contained four copies
of the 94-bp wild-type or mutant PAI-1 promoter sequences (
740/
647) inserted upstream of the TATA box in the SV40 early promoter were generated (Fig. 4A). The
ability of these promoter sequences to mediate TGF
-induced
activation of luciferase gene expression was then examined in Hep3B
cells (Fig. 4B). Consistent with the DNA binding results,
mutation of site II only slightly reduced TGF
-induced
transcriptional activation, from 25- to 15-fold (M1, Fig.
4B). This decrease in the fold of transactivation is mostly due to a reproducible increase in the basal level of transcription in
the absence of TGF
1, suggesting that site II may be occupied by an
inhibitory factor in the absence of the ligand. Mutation of site I
(SBE-like site) markedly impaired the ability of this promoter region
to mediate TGF
1-induced transactivation (M2, Fig. 4B).
Mutation of both site I and site II greatly reduced binding of Smad3
and Smad4, and the mutant promoter was unable to respond to TGF
1
(M3, Fig. 4B). These results indicate that the two
Smad-binding sites in the PAI-1 promoter we identified were necessary
for TGF
-induced transcriptional activation.

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Fig. 4.
The two Smad-binding sites in the human PAI-1
promoter are required for TGF -induced, Smad3-
and Smad4-dependent transcriptional activation.
A, luciferase reporter constructs used in the
transactivation experiments contain four copies of the wild-type or
mutant PAI-1 promoter DNA sequences. B, Hep3B cells were
transfected with 2 µg of luciferase reporter constructs listed in
A and stimulated with 100 pM TGF 1 for 16 h. Luciferase activity was measured as described under "Experimental
Procedures." The experiment was repeated 5 times and one
representative set of results are shown. C, Hep3B cells were
co-transfected with the 4x wild-type luciferase reporter construct and
various Flag-tagged Smad proteins. Luciferase activity was measured
48 h after transfection in the absence of TGF 1. The addition of
TGF 1 did not further increase the level of transactivation.
D, Hep3B cells were co-transfected with wild-type or mutant
4x wild-type luciferase reporter constructs together with Flag-Smad3
and Flag-Smad4. Luciferase activity was assayed 48 h after
transfection in the absence of TGF 1.
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To examine whether transactivation mediated by the two Smad-binding
sites are dependent on Smad3 and Smad4, we co-expressed various Smad
proteins together with the luciferase reporter construct containing
four copies of the wild-type PAI-1 promoter sequence. Consistent with
their DNA binding activity, overexpression of Smad3 and Smad4 in Hep3B
cells activated PAI-1 promoter activity (Fig. 4C). Smad2
failed to bind to this promoter sequence (data not shown), and it did
not activate PAI-1 transcription (Fig. 4C). Since the N
domains of Smad3 and Smad4 are responsible for DNA binding, we next
examined whether they can activate transcription from this PAI-1
promoter region. Expression of Smad3N or Smad4N alone (data not shown),
or both together (Fig. 4C), activated PAI-1 transcription to
the same extent as did the full-length Smad3 and Smad4. In contrast,
expression of the COOH-terminal domains of Smad3 and Smad4 did not
activate PAI-1 transcription, consistent with the lack of DNA binding
activity by these C domains (Fig. 2).
To determine whether Smad3- and Smad4-induced transactivation is
dependent on the two Smad-binding sites, we co-expressed Smad3 and
Smad4 with various mutant PAI-1 promoters in Hep3B cells (Fig.
4D). Mutation of site II alone resulted in a 60% reduction in transactivation while alteration of site I, or both site I and site
II abolished the ability of Smad3 and Smad4 to activate PAI-1
transcription (Fig. 4D). Therefore, direct binding of Smad3 and Smad4 to the two Smad-binding sites in the PAI-1 promoter through
their NH2-terminal domains is required for transcriptional activation.
Homoligomerization of Smad3 Enhances Binding of Smad3 to the Two
Smad-binding Sites--
Upon ligand stimulation, Smad3 can be
phosphorylated by the activated T
RI and form homo- as well as
heteroligomers. To investigate whether activation by T
RI affects the
DNA binding activity of Smad3, Flag-Smad3 was co-transfected with or
without the constitutively active T
RI(T204D), and the ability of
Smad3 to bind to 32P-labeled probe A was examined by EMSA.
Co-transfection of the active T
RI significantly enhanced binding of
Smad3 to DNA (lanes 1 and 2, Fig.
5A), suggesting that
phosphorylation and oligomerization of Smad3 may increase the affinity
of Smad3 for DNA. To investigate the effects of homo- and
heteroligomerization of Smad proteins on DNA binding, Smad3 mutants
containing point mutations changing Arg-268 to Cys, Val-277 to Asp, or
Asp-408 to Glu were generated. These amino acid residues localize at
the trimer interface and may mediate homo- and heteroligomerization and
are conserved among Smad2, Smad3, and Smad4 (46). Point mutations of
these three residues in Smad2 and Smad4 were originally identified in
human cancer and may abolish both homo- and heteroligomerization of Smad proteins (46).

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Fig. 5.
The effects of homo- and heteroligomerization
of Smad3 and Smad4 on binding to the two Smad-binding sites.
A, EMSA assay was performed with 32P-labeled
probe A and an equal amount of wild-type and mutant Smad3 isolated from
singly (lane 1) or co-transfected 293T cells (lanes
2-5), and wild-type and mutant Smad4 (lanes 6-10).
B, homo- and heteroligomerization of Smad3 and Smad4
mutants. HA- or Flag-tagged Smad3 or Smad4 proteins were isolated by
immunoprecipitation from co-transfected 293T cells and analyzed by
Western blotting as indicated. C, Hep3B cells were
co-transfected with the 4x wild-type luciferase reporter construct and
wild-type or mutant Smad3 (left panel) and Smad4
(right panel). Luciferase activity was measured 48 h
after transfection.
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The ability of Smad3 containing the Arg (R268C), Val (V277D), or Asp
(D408E) mutation to homo- and heteroligomerize was examined first by
co-immunoprecipitation experiments (Fig. 5B). HA-tagged wild-type or mutant Smad3 was co-transfected together with
T
RI(T204D) and Flag-Smad3 (for homoligomerization) or Flag-Smad4
(for heteroligomerization) into 293T cells. Following
immunoprecipitation with an anti-HA antibody, the presence of
Flag-tagged Smad3 or Smad4 in the immune complex was detected by
Western blotting using an anti-Flag antibody. As expected, all three
Smad3 mutants were defective in heteroligomerization (upper left
panel, Fig. 5B). Their ability to homoligomerize was also markedly impaired (lower left panel, Fig.
5B). The ability of these Smad3 mutants to bind DNA was
tested next by EMSA with labeled probe A (Fig. 5A). Mutant
Smad3 proteins isolated from co-transfected 293T cells (lanes
3-5, Fig. 5A) exhibited much weaker binding to probe A
than wild-type Smad3 (lane 2, Fig. 5A), suggesting that the wild-type DNA-Smad3 complex is due to the activity
of the Smad3 homoligomers and that oligomerization of Smad3 greatly
increases the affinity of Smad3 for DNA. Consistent with the decreased
binding activity, the ability of the R268C and V277D mutants to mediate
transcriptional activation was also reduced (Fig. 5C).
Similar Arg, Val, and Asp mutations were introduced into Smad4. These
mutations were found previously to abolish both homo- as well as
heteroligomerization of the Smad4 C domain (15). As expected, we found
that these mutant Smad4 were defective in heteroligomerization with
Smad3 (right panel, Fig. 5B). However, they did
not disrupt homoligomerization of the full-length Smad4 (Fig.
5B), probably because the N domain of Smad4 can also mediate oligomerization (15). These mutant Smad4 proteins bound to probe A in a
manner indistinguishable from that of wild-type Smad4 (lanes 6-10, Fig. 5A), suggesting that heterotrimerization of
Smad4 with Smad3 is not essential for Smad4 to bind DNA. However, since
these mutants still form homoligomers, it is not clear whether
homoligomerization is required for Smad4 to bind DNA. In transcription
assays, these two mutants displayed a slightly reduced transactivation
activity than did the wild-type Smad4 (Fig. 5C). Therefore,
heteroligomerization of Smad4 with Smad3 does not affect direct binding
to DNA, but may be required for optimal transactivation in
vivo.
A Mutation in Smad4, R100T, Identified Originally in a Human
Pancreatic Carcinoma Abolished the DNA Binding Activity of
Smad4--
Mutations in Smad4 have been detected in many types of
human cancer. One mutation, R100T, found in a pancreatic carcinoma, is
located in the N domain and was reported to inactivate Smad4 function
through an autoinhibitory mechanism (47). We asked whether the R100T
mutation affects the ability of Smad4 to bind DNA. As shown in Fig.
6A, Flag-tagged, full-length
as well as the NH2-terminal domain of Smad4 containing the
R100T mutation isolated from transfected 293T cells failed to bind DNA
(lanes 2 and 4, Fig. 6A). The lack of
DNA binding could be due to disruption of the Smad4 DNA binding
activity by the point mutation or due to the autoinhibitory effect,
e.g. blocking of the Smad4 N domain by the interacting Smad4
C domain or by other interacting molecules present in the transfected
293T cells. To distinguish between these two possibilities, the
recombinant N domain of Smad4(R100T) was purified from E. coli and was found to display no DNA binding activity (lane
8, Fig. 6A), suggesting that this point mutation directly destroyed the DNA binding activity of Smad4. Consistent with
the DNA binding data, Smad4(R100T) failed to activate transcription from the PAI-1 promoter (Fig. 5C). Our results were
consistent with those reported by Song et al. (37) and Kim
et al. (30) that the R100T mutation destroys the DNA binding
activity of Smad4, but did not agree with that reported by Shi et
al. (34).

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Fig. 6.
The point mutation in Smad4, R100T, abolished
the ability of Smad4 to bind DNA. A, EMSA assay was
performed with 32P-labeled probe A and an equal amount (200 ng) of full-length and truncated Smad4 and Smad4(R100T) isolated either
from transfected 293T cells (Flag-tagged, lanes 1-4) or
from E. coli (lanes 5-8). B, Western
blotting analysis of Smad4 proteins using anti-Smad4 antibody.
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DISCUSSION |
We have identified two major Smad-binding sites in the major
TGF
-responsive region of the PAI-1 promoter which interact with Smad3 or Smad4 in a cooperative manner and are required for
transcriptional activation in a TGF
-induced, Smad3 and
Smad4-dependent manner. One of the sites (site I) contains
a SBE-like element (GTCTGGAC) with a perfect Smad box and a
single base pair mismatch (underlined nucleotide). This single
nucleotide mismatch results in a significant decrease in Smad binding
(33). Interestingly, the second Smad-binding site we identified (site
II) is part of an AP1-like motif. This AP1-like site has been suggested
to play a role in mediating TGF
-induced transactivation of both the
PAI-1 gene (35) and type I collagen gene (44). The Smad box in this
site contains a mismatch that should decrease its affinity for the Smad
proteins significantly (34). Indeed, compared with site I, the binding
of Smad3 and Smad4 to site II was markedly weakened. Although the
affinity of Smads to either site I or site II alone is not optimal, the cooperative effect mediated by multiple sites present in close proximity may increase the affinity significantly. Interestingly, although another degenerate Smad box (GTAT) is present in another AP1-like site located 3' to site II, this sequence did not mediate Smad
binding. Another degenerate Smad box (AGCC) located immediately 3' to
site II was not recognized by Smad proteins either, since a mutant
probe (M4) containing this sequence but lacking site II did not bind
Smad proteins (Fig. 3A). Therefore, sequences surrounding
the core Smad box also play an important role in the recognition of
Smad proteins.
Dennler et al. (32) have identified three CAGA containing
sequences in the PAI-1 promoter that mediate binding to Smad3N and
Smad4, and are required for TGF
-induced transcriptional activation of the PAI-1 promoter. One of the three CAGA sequences is located in
this major TGF
responsive region of the PAI-1 promoter. Song et al. (37) also reported a 12-bp sequence overlapping with the CAGA sequence that mediates binding of Smad3 and Smad4. Compared with the two Smad-binding sites we identified, however, Smad binding mediated by these motifs is much weaker. Therefore, under our experimental conditions, the major Smad binding activity is mediated by
sites I and II.
Our result that the two Smad-binding sites acted cooperatively to
mediate binding of Smad4 and to a lesser extent, Smad3, differs from
the report by Shi et al. (34) that two repeats of the Smad
box did not display cooperativity in mediating binding of the MH1
domain of Smad3. The apparent difference could be due to the different
Smad protein preparations used in the EMSA assays. First of all, only
part of the MH1 domain of Smad3 (amino acid residues 1-145) was tested
by Shi et al. (34) while our experiments used full-length
Smad4 and Smad3. We found that the cooperativity of the two sites in
mediating binding of Smads is more prominent with full-length Smad
proteins and much less so with the MH1 domains alone (Fig. 2,
B and C, and data not shown). This cooperativity requires the C domains of the Smads and could be due to
homoligomerization of full-length Smad proteins. Consistent with this
hypothesis, disruption of oligomerization of Smad3 by mutation resulted
in a decreased affinity for the two sites and a reduced mobility shift.
These mutants also exhibited a much decreased ability to activate transcription.
Direct DNA Binding by Smads Is Critical for Transcriptional
Activation by TGF
--
Although several reports have demonstrated
that bacterially produced Smad3 and Smad4 can bind DNA directly
(30-33, 49), it is not clear whether this binding activity is critical
for transcriptional activation. Our findings that the
NH2-terminal domains of Smad3 and Smad4 directly bound to
two adjacent DNA sequences in the PAI-1 promoter and activated
transcription in response to TGF
directly link the DNA binding
activity of Smads with their ability to transactivate. The C domains of
Smad3 and Smad4 did not bind DNA, and failed to activate transcription
from the two Smad-binding sites (Fig. 4C). Our result is
consistent with a previous report that the N domain of Smad4 possessed
transactivation activity both in yeast and in mammalian cells (15). The
N domain of Smad3 was found to interact with c-Jun (27) and the N
domain of Smad4 was shown to interact with CBP/p300 (28). These
interactions may play a role in transactivation by the N domains of
Smad proteins. The C domains of Smad3 and Smad4 have been shown by many
studies to contain transactivation activity. The C domain of Smad3 has been shown to interact with general transcription factors including Sp1
(26), AP1 (27), and CBP/p300 (28, 29). The Smad3 C domain may cooperate
with these proteins to activate transcription. The N domains of Smad
proteins could activate transcription by a mechanism different from
that used by the C domains.
Multiple mechanisms may be involved in TGF
-induced activation of
gene expression. The PAI-1 promoter contains many regions that can
mediate TGF
responses. In addition to the three CAGA sequences (32)
and the two Smad-binding sites we identified here, an E box region that
interacts with the transcription factor TFE3 and Smad3 was also shown
to mediate TGF
responses (48). Therefore, it is possible that Smad
proteins may be present in multiple transcription complexes on
different regions of the promoter and activate transcription through
different mechanisms.
We have shown that the two Smad-binding sites in the PAI-1 promoter are
necessary for TGF
-induced transactivation. Consistent with the DNA
binding activity, mutation of either one of the two sites decreased
TGF
-induced transcriptional activation, while mutation of both sites
abolished this activity completely. Although the two Smad-binding sites
act cooperatively to mediate binding of Smad proteins, no cooperativity
in transactivation is apparent. It is possible that binding of Smad
proteins to DNA is not the rate-limiting step during transcriptional
activation, and interaction of Smad proteins with other cellular
co-factors may stabilize their binding to DNA in vivo.
We have found that a point mutation in Smad4, R100T, originally
identified in a human pancreatic carcinoma, abolished DNA binding
activity of Smad4, and this mutant Smad4 cannot mediate activation of
PAI-1 transcription. Since this residue is not directly involved in
contacting DNA (34), R100T mutation is likely to affect the
conformation of the DNA-binding domain of Smad4, rendering it incapable
of binding to DNA. Partial proteolytic analysis revealed changes in
sensitivity to protease digestion (data not shown), suggesting that the
R100T mutation caused conformational changes in Smad4. Our result is
consistent with those reported by Song et al. (37) and Kim
et al. (30), but not with that by Shi et al.
(34). Taken together, these results provide a good correlation between
DNA binding activity and the ability to transactivate by Smads.