From the Department of Developmental Genetics,
Groningen Biomolecular Sciences and Biotechnology Institute, P. O. Box
14, 9750 AA Haren, The Netherlands and the ¶ Ludwig Institute for
Cancer Research, Box 595, Biomedical Center, S-75,
24 Uppsala, Sweden
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ABSTRACT |
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Smad proteins have been identified as mediators
of intracellular signal transduction by members of the transforming
growth factor- (TGF-
) superfamily, which affect cell
proliferation, differentiation, as well as pattern formation during
early vertebrate development. Following receptor activation, Smads are
assembled into heteromeric complexes consisting of a pathway-restricted Smad and the common Smad4 that are subsequently translocated into the
nucleus where they are thought to play an important role in gene
transcription. Here we report the identification of Smad Binding
Elements (SBEs) composed of the sequence CAGACA in the promoter of the
JunB gene, an immediate early gene that is potently induced
by TGF-
, activin, and bone morphogenetic protein (BMP) 2. Two
JunB SBEs are arranged as an inverted repeat that is
transactivated in response to Smad3 and Smad4 co-overexpression and
shows inducible binding of a Smad3- and Smad4-containing complex in
nuclear extracts from TGF-
-treated cells. Bacterial-expressed Smad
proteins bind directly to the SBE. Multimerization of the SBE creates a
powerful TGF-
-inducible enhancer that is also responsive to activin
and BMPs. The identification of the sequence CAGACA as a direct binding site for Smad proteins will facilitate the identification of regulatory elements in genes that are activated by members of the TGF-
superfamily.
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INTRODUCTION |
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The product of the JunB gene is a member of the AP-11 family of transcription factors that activate transcription by binding to TPA response elements (TREs) within the promoter of target genes (1). AP-1 components are immediate early gene products whose expression is rapidly induced by a variety of extracellular stimuli and are encoded by the Fos and Jun families of genes that have been shown to be involved in growth control and differentiation (2). JunB differs in biological properties from its homologs and appears to be a negative regulator of AP-1 function (3). This functional difference is because of a small number of amino acid changes in its DNA binding and dimerization motifs compared with the corresponding c-Jun sequences, as well as to differences in phosphorylation status in response to mitogenic stimulation (4).
The action of JunB as a negative regulator of TRE response
elements is consistent with its induction by negative regulators of
cell growth including transforming growth factor- (TGF-
) as well
as the structurally and functionally related factors activin and bone
morphogenetic protein (BMP) 2/4 (5-8). In this respect, JunB is a member of the group of genes that are known to be
induced in response to TGF-
stimulation, which further include the
cyclin-dependent inhibitors (CDI) p15 and p21 (9, 10) and
the plasminogen activator inhibitor (PAI-1) gene (11) that control, in
part, cell cycle progression and extracellular matrix remodeling in response to TGF-
, respectively. However, it is presently unclear whether the induction of these genes by TGF-
and related factors involves a direct mechanism.
TGF- family members exert their cellular effects (12, 13) by
binding to transmembrane receptors that possess serine/threonine kinase
activity (14). Upon ligand binding, a heteromeric receptor complex
consisting of two type II and two type I receptors is formed. Within
the complex, the type I receptor is phosphorylated and activated by the
type II receptor constitutively active kinase. Genetic studies in
Drosophila melanogaster and Caenorhabditis elegans have recently led to the identification of a conserved family of proteins termed Smads that play an important role in intracellular signal transduction of serine/threonine kinase receptors (15). At present, at least nine family members have been identified in
vertebrates. Smads are 40-62-kDa proteins with N- and C-terminal homology domains (MH1 and MH2) connected by a proline-rich linker. Smad1, Smad5, and presumably MADH6/Smad9 associate with and are phosphorylated after BMP-mediated BMP-RI and ActR-I activation, whereas
Smad2 and Smad3 are phosphorylated after TGF-
R-I and ActR-IB
activation. Following phosphorylation, which occurs at a conserved SSXS
motif at the extreme C termini, these pathways-restricted Smads form
heteromeric complexes with the common mediator SMAD4 and translocate to
the nucleus to regulate gene transcription. Phosphorylation of the
proline linker by growth factor-activated Erk mitogen-activated protein
kinase was recently shown to have an inhibitory effect on BMP-induced
nuclear accumulation of Smad1 (16). Smad6 and Smad7 are structurally
and functionally distinct from other Smads in that they lack an MH1
domain and act opposite to the pathway-restricted and common mediator
Smads; they block the activation of pathway-restricted signals by
competition for receptor association or by preventing heteromeric
complex formation between pathway-restricted and common mediator Smads
(17-19).
Support for a role of Smads as transcription factors has been obtained
from a number of studies. The C-terminal domains of Smad1 and Smad4
have transactivation activity when fused to the Gal4-DNA binding domain
in a Gal4-reporter transactivation assay (20, 21). Smad2 and Smad4
together with Fast-1, a winged-helix DNA binding protein, associate
into activin response factor (ARF) that binds to the activin response
element of the Xenopus laevis Mix.2 promoter (22, 23).
TGF- as well as Smad3 and Smad4 overexpression transactivate the
PAI-1 and p3TP-lux promoters (24), which has been attributed to
potentiation of AP-1-dependent transcription activation
(25). By contrast, Drosophila Mad domain binds a G+C-rich
sequence in the Drosophila vestigial quadrant enhancer (26).
However, these investigations have not been conclusive regarding the
role of Smads in transcriptional activation, as well as regarding the
sequences with which they interact.
In this report, we have investigated the transcriptional regulation of
the immediate early gene JunB by TGF-. Transient
overexpression of Smad3 and Smad4 with various JunB promoter
constructs led to the identification of a region in the upstream part
of the promoter that is both Smad-transactivated and shows
TGF-
-induced binding of a Smad-containing complex. Further
characterization led to the identification of an inverted
hexanucleotide repeated sequence binding a TGF-
-induced DNA binding
activity. Multimerization of this sequence created a powerful
TGF-
-inducible enhancer. Implications of these findings for Smad DNA
binding and transcriptional activation will be discussed.
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EXPERIMENTAL PROCEDURES |
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Construction of Plasmids--
An 8-kilobase EcoRI
fragment was isolated from the Balb/c mouse genomic JunB
clone 31 which has been described previously (27). All sequences
upstream from the initiator ATG were cloned in the pGL3-basic reporter
vector (Promega). For cloning purposes, the ATG was converted to ATC by
PCR (pJB1). A deletion series was constructed by restriction digestion
of the pJB1 plasmid with EcoRI-PinAI (pJB2),
SacI (pJB3), SacI-BssHII (pJB4), or
SacI-SacII (pJB5) and subsequent self-ligation. A
minimal promoter construct (pGL3ti) was made from pGL3-basic by
inserting oligonucleotides carrying the adenovirus major late promoter
TATA box (AdTu, gatctGGGGCTATAAAAGGGGGTAGGGGgagct, and AdTl,
cCCCCTACCCCCTTTTATAGCCCCa) and the mouse terminal deoxynucleotidyl transferase gene initiator sequence (Tiu, cGCCCTCATTCTGGAGACAg, and
Til, gatccTGTCTCCAGAATGAGGGCgagct) in the BglII site. A
deletion series (pJB11-17) was constructed as follows: an
Asp718-BseAI (
3004/
1561) fragment from pJB3
was inserted into the Asp718-XmaCI sites from
pGL3ti (pJB11), a T4 DNA polymerase-blunted
SacI-BamHI fragment from pJB3 was inserted in the
SmaI site of pGL3ti (pJB12), a
BamHI-BglII fragment from pJB11 was inserted into
the BglII site of pGL3ti (pJB13), pJB12 was
restriction-digested with Asp718-PvuII or
PvuII-BglII and subsequently blunted and
self-ligated (pJB14 and pJB15, respectively), pJB15 was restriction
digested with Asp718-MluNI and subsequently
blunted and self-ligated (pJB17), and pJB11 was digested with
MluNI and BglII and subsequently blunted and
self-ligated (pJB16). For fine mapping of the Smad3 and Smad4 response
site in the
2908/
2611 region, pJB15 was used as template for PCR
reactions with the Til oligonucleotide and the upper strand of
oligonucleotide D (see "Electrophoretic Mobility Shift Assays"). The PCR product was restriction-digested with SacI and
ligated into an Asp718-digested and blunted,
SacI-digested pGL3ti vector (pGL3ti-DPv). pGL3ti-PsPv and
pGL3ti-AfPv were constructed by digestion of pJB15 with
Asp718-PstI or
Asp718-AflII, blunting and self-ligation. The
internal deletion series was constructed as follows: a
PstI/PvuII fragment (
2762/
2611) from pJB12
was cloned into the PstI and EcoRV sites of
pBluescript SKII- (pSK-PsPv). Next, pJB15 was used as template for PCR
reactions with the upper strand of oligonucleotide A and the lower
strands of oligonucleotides C, D, and G. The PCR products were
cloned into the SmaI site of pSK-PsPv. The resulting
plasmids were restriction-digested with XbaI and
SalI, and the fusion fragments were inserted into
NheI-XhoI-opened pGL3ti.
pGL3ti-(SBE)4 was constructed by inserting two WT
oligonucleotides (see "Electrophoretic Mobility Shift Assays") into
the XhoI site of pGL3ti. All constucts were verified by
sequencing. The nucleotide sequence of the
2908/
2611
MluNI/PvuII Smad-responsive region appeared to
differ at several positions from a previously deposited JunB
genomic sequence (GenBank accession number U20735). The nucleotide
sequence of this region as determined by us has been deposited in the
EBI Data Bank (accession number AJ004891). Smad expression plasmids
were constructed as follows: pSG5-XMAD1 and pSG5-XMAD2 were created by
inserting EcoRI fragments from pSP64TEN-DOT1 and -DOT2 into
EcoRI-opened pSG5, pSG5-hSmad3f was created by inserting a
blunted BamHI-SalI fragment from pRK5-hSmad3f into blunted BamHI-opened pSG5, and pSG5-hSmad4 was created
by inserting a blunted BamHI-EcoRI fragment from
pDPC-wt3 into blunted EcoRI-BamHI-opened pSG5.
The expression plasmid for Flag-Smad1, Smad2, Myc-Smad3, and Smad4 were
described previously (28).
Cell Culture and Transient Transfections--
P19EC embryonal
carcinoma cells were maintained in a 1:1 mixture of Dulbecco's
modified Eagle's medium (DMEM) and Ham's F12 medium supplemented with
7.5% fetal bovine serum (Integro, Zaandam, The Netherlands). NIH3T3
embryonic fibroblast cells were maintained in DMEM supplemented with
10% newborn calf serum (Life Technologies, Inc.). HaCaT human
keratinocytes, HepG2 human hepatocellular carcinoma cells, MDA-MB468
(obtained from ATCC) breast cancer cells and Mv1Lu mink lung epithelial
cells were grown in DMEM medium supplemented with 10% fetal calf
serum. All media were further supplemented with 100 IU/ml penicillin,
100 µg/ml streptomycin, 2 mM L-glutamine, and
1× MEM nonessential amino acids. For transient transfection, cells
were seeded at 5 × 104 (P19) or 2.5 × 105 (HepG2 and MDA-MB468) cells per well of a 6-well tissue
culture cluster. The next day, cells were transiently transfected using the calcium phosphate co-precipitation method. After 24 h, the medium was changed to DMEM supplemented with 0.3% fetal calf serum. Simultaneously, the cells were stimulated with 10 ng/ml TGF-1 (R&D
Systems), 20 ng/ml activin A, 100 ng/ml Osteogenic Protein-1 (OP-1),
100 ng/ml BMP2, or 200 ng/ml Growth/Differentiation Factor 5 (GDF5) for
16 h. The cells were lysed with reporter lysis buffer (Promega),
followed by measurement of luciferase activity using luciferase assay
system (Promega). In all transfections,
-galactosidase expression
plasmids (pDM2LacZ (29) or pCH110, Pharmacia) served as internal
controls to normalize the luciferase activity.
-Galactosidase activity was quantified in 100 mM
Na2HPO4/NaH2PO4, 1 mM MgCl2, 100 mM 2-mercaptoethanol,
and 0.67 mg/ml O-nitrophenylgalactopyranoside. Each
transfection was carried out in triplicate and repeated at least
twice.
Electrophoretic Mobility Shift Assays--
Nuclear extracts were
prepared from Mv1Lu cells using a modified Dignam protocol as described
previously (30). Oligonucleotides and dephosphorylated restriction
fragments were labeled with [-32P]ATP and T4
polynucleotide kinase. Oligonucleotides used in electrophoretic mobility shift assay experiments are shown in Scheme
1. Binding reactions contained 4 µg of
nuclear extract, 100 mM KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 20% glycerol, 20 mM HEPES,
pH 7.9, 100 ng of poly(dI-dC) and approximately 10.000 cpm labeled
probe and 500-fold molar excess of competitor oligonucleotide where appropriate. Protein binding was allowed to proceed for 30 min at room
temperature. Then 20% Ficoll was added to the reactions, and samples
were immediately loaded onto 4.5 or 5% polyacrylamide gels containing
0.5× TBE. Smad3 and Smad4 antibodies (DHQ and HPP, respectively) (28)
were added undiluted at 0.5 µl, after proteins were allowed to bind
the probe for 15 min. Samples were then incubated for 15 min before
loading onto the gel.
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RESULTS |
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Identification of a Smad-responsive Region in the JunB
Promoter--
Previously, we have shown that the immediate early gene
JunB is a direct target for transcription activation in
response to activation of signal transduction by TGF- (5). To
investigate the mechanism of transactivation of the JunB
promoter by TGF-
, a JunB promoter-luciferase reporter
construct was made containing the JunB TATA box and
transcription start site, the complete 5'-untranslated region as well
as approximately 6.4 kilobases of promoter upstream sequences (pJB1).
Transient transfection of this construct in NIH3T3, Mv1Lu, and HepG2
cells and treatment of the cells with TGF-
did not result in a
significant increase in activation of the luciferase reporter construct
over uninduced levels after normalization with
-galactosidase
activities from cotransfected control LacZ expression plasmid (data not
shown). Recently, we and others have shown that transiently
overexpressed Smad proteins transactivate target genes in a
ligand-independent manner (24, 28). We therefore cotransfected pJB1
with plasmids expressing individual Smads or combinations of each
pathway-restricted Smad and Smad4 into NIH3T3 cells (Fig.
1A). A 3- to 5-fold activation of the reporter construct was observed when Smads 1, 2, and 3 were
co-expressed with Smad4 while individual Smads did not significantly activate the reporter. Similar results were obtained using P19 embryonal carcinoma (EC) cells and HepG2 cells. We localized the Smad-responsive region in the JunB promoter by transfecting
cells with a series of deletion constructs along with Smad3 and Smad4, which is the strongest activating combination (see Fig. 1A).
This analysis showed that the Smad-responsive region is located between nucleotides
3004 and
1534; whereas pJB3 has Smad inducibility very
similar to pJB1, this response was lost for the JB4 deletion construct
(Fig. 1B). A nested set of restriction fragments was derived
from this Smad-responsive region and cloned in front of a heterologous
minimal promoter fused to the luciferase gene. Cotransfection of these
constructs with Smad3 and Smad4 expression plasmids allowed
localization of a Smad-responsive region to between nucleotides
2908
and
2611 (Fig. 1C). The position of the Smad-responsive element within the
2908/
2611 region was determined using a series of progressive 5' and internally deleted constructs. Deletion of
sequences upstream from nt
2813 did not affect Smad responsiveness while additional deletion of 26 bp (to nt
2787) abrogated
inducibility. This analysis was complemented by a series of deletions
of sequences located between nt
2908 and
2762. Exclusion of
sequences upstream from nt
2792 abrogated transactivation by Smad3
and Smad4 (Fig. 1D). This analysis therefore defines
the 22-base pair region between
2813 and
2792 as that minimally
required for transactivation by Smad3 and Smad4. Interestingly,
the 22-bp sequence contains a perfect 7-bp inverted repeat
(CAGACAGtCTGTCTG).
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JunB Promoter Fragments Bind TGF--induced Complex Containing
Smad3 and Smad4--
To investigate whether TGF-
induces binding of
nuclear proteins to the Smad-responsive region, we succesively
incubated labeled probes containing nt
2908 to
2611, nt
2908 to
2788, and nt
2813 to
2792 with nuclear extracts from
TGF-
-treated or -untreated Mv1Lu, HaCaT, and NIH3T3 cells cells
(Fig. 2). Electrophoretic mobility shift
assay experiments showed that extracts from TGF-
-treated cells
contain an induced DNA binding activity that migrates with a lower
mobility than that of a constitutively expressed binding entity. These
results suggest that TGF-
activates the endogenous JunB
gene by inducing binding of a nuclear factor to a JunB
promoter distal element located between nucleotides
2813 and
2792.
This fragment correlates with the minimal region required for
transactivation by Smad3 and Smad4, suggesting that Smads may form part
of nuclear complexes that bind to the 22-bp JunB promoter
sequence. To analyze whether Smads are present in nuclear complexes
that bind to the 22-bp JunB promoter sequence, antisera
directed against Smad3 or Smad4 were added to the binding reactions of
the 22-bp JunB probe with nuclear extracts (Fig.
2C). Both Smad3 and Smad4 antisera supershifted the slowest
migrating TGF-
-induced complex. The Smad3 antiserum was more
effective than the Smad4 antiserum, which might be due to intrinsic
higher affinity of Smad3 antiserum versus Smad4 antiserum.
Both antisera produced a supershifted band with extracts from uninduced
cells. Possibly, these complexes were formed by stabilization by the
antisera of the interaction of contaminating cytoplasmic Smads with the
probe. Neither Smad1, 2, or 5 antiserum produced supershifted bands
(data not shown). These results indicate that TGF-
induces nuclear
translocation of activated Smad3 and Smad4 and their subsequent binding
to a defined region in the JunB promoter.
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Smad Proteins Bind Directly to CAGACA Elements--
Having
identified Smad3 and Smad4 as components of the TGF--induced
complexes, we next investigated whether Smad proteins directly bind the
Smad-responsive fragment. We produced C-terminal truncated (
MH2) and
full-length Smads 1-5 as GST fusion proteins and analyzed these for
their ability to bind the 120-bp MluNI/AflII (
2908/
2788) fragment (Fig.
3A). Smad3
MH2 and
Smad4
MH2 strongly interacted with the probe while binding of
C-terminal truncated Smads 1, 2, and 5 to the 120-bp fragment was below
the threshold of detection. Binding of full-length Smad4, but not Smad3
fusion protein, was observed, indicating that Smad3 requires a
modification or a conformational change to demonstrate its DNA binding
potential. These results show that Smad3 and Smad4 bind directly to
their target sequence in the 120-bp JunB promoter
fragment.
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The Central GAC Nucleotides in the SBE Are Most Important for Smad3
and Smad4 Binding--
To identify bases in the SBE that are important
for Smad3 and Smad4 binding, we tested the efficiency of binding of
Smad3 and Smad4 to direct repeats of the SBE or mutated versions
thereof in which each repeat carried a different substitution of one of the base pairs of the 7 bp of the inverted dimeric SBE (Fig.
4A). The direct repeat and the
inverted repeat bound Smad3 and Smad4 equally well (data not shown).
The order of binding strength of Smad proteins to the oligonucleotides
was as follows: WT = M7 > M6 > M1 > M2 > M4 > M3 = M5. Complementary experiments in which the binding
of GST-Smad proteins was challenged by incubation with excess unlabeled
wild type or mutant oligonucleotides as competitors yielded results
with similar affinity differences between wild type and mutant probes
(Fig. 4B). Essentially the same results were obtained for
binding of proteins from nuclear extracts, which include Smad3 and
Smad4, isolated from TGF--induced cells to the 22-bp inverted
repeat-containing probe (data not shown). When we tested the Smad
binding to a probe containing four directly repeated SBEs, we found
that Smad3
MH2, Smad4
MH2, and full-length Smad4 bound with much
higher affinity than two repeats (Fig. 4C). The same results
were obtained when the SBEs were present as two indirect repeats (data
not shown). In addition, weak binding of full-length Smad3 was now
observed. We could specifically compete the Smad3 or Smad4 binding with
wild type oligonucleotide, but not with a mutant version in which the
G3 and C5 were substituted, and no binding to mutated probe was
observed (Fig. 4C). This analysis defines the central
nucleotides GAC in the SBE as most important for binding Smad proteins
and the TGF-
-induced complex, while the flanking nucleotides
contribute less to the binding and the most 3' nucleotide (G7) is not
essential.
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The SBE Is a TGF- Response Element--
To investigate whether
the SBE can confer inducibility to TGF-
, we inserted the SBE in
different copy number in front of a minimal promoter and tested these
constructs in HepG2 cells. We found that two copies were insufficient
for significant induction of luciferase activity, but that with four
copies a strong response was obtained (Fig.
5A). Therefore,
multimerization of the SBE is sufficient to confer TGF-
inducibility
to a minimal promoter. When the (SBE)4 reporter was
cotransfected with Smads, we found that Smad3 alone, but not Smad2 or
Smad4 alone, had a slight ligand-independent effect. The
TGF-
-induced effect was enhanced by cotransfecting the Smad3 and
Smad4 combination, but not with the Smad2 and Smad4 combination.
Highest TGF-
-induced transcriptional response was obtained when all
three Smads were cotransfected (Fig. 5A).
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Smads Participate in TGF--induced SBE-mediated
Transcription--
TGF-
failed to induce the (SBE)4
reporter in MDA-MB468 cells that lack the Smad4 gene (31)
(Fig. 5B). As the TGF-
response was rescued by Smad4
transfection, Smad4 appears to be required for the TGF-
-induced,
SBE-mediated transcriptional response. Further transfection studies
showed that Smad3 and Smad4, but not Smad2 and Smad4, cooperated in the
SBE-mediated transcriptional response in these cells, which was readily
observed in the absence of ligand. In fact, cotransfection of Smad3 and
Smad4 resulted in a level of reporter activation over which no dramatic
TGF-
-dependent respone was observed. Cotransfection of
the reporter plasmid into HepG2 cells along with increasing amounts of
a Smad7 expressing plasmid showed a dose-dependent decrease
in TGF-
-induced SBE-mediated transcriptional response (data not
shown). Taken together, the results indicate that Smad3 and Smad4 are
directly involved in the T
R-I-mediated activation of the
JunB promoter-derived SBE.
SBE Is a Response Element for Other Members of the TGF-
Family--
Activin and BMP2 have been shown to induce JunB
mRNA expression (7, 8). To test whether the SBE can confer
inducibility to activin and BMP, we transfected HepG2 cells with the
TGF-
-responsive (SBE)4 construct and treated the
transfected cells with either OP-1 (also termed BMP-7), activin, or
TGF-
(Fig. 5C). All three ligands activate the SBE
reporter albeit with different efficiencies. To corroborate these
findings, we transfected Smad4-negative MDA-MB468 cells with the CAGACA
reporter plasmid with or without the Smad4 expression plasmid and
tested the transfected cells for responsiveness to TGF-
, activin,
OP-1, BMP2, and GDF5 (Fig. 5D). In the absence of Smad4, the
reporter construct was virtually unresponsive to any of the ligands.
Cotransfection of Smad4 resulted in a slight activation of the
reporter. However, treatment of the Smad4 cotransfected cells with the
various members of the TGF-
superfamily showed that OP-1 and TGF-
strongly activated the (SBE)4 construct, whereas treatment
with activin was without effect, which may indicate that our MDA-MB468
cells lack functional activin receptors. Furthermore, BMP2 and GDF5,
which bind the BMP type IB receptor (BMPR-IB) (32) also strongly
activated the (SBE)4. In agreement with these findings, constitutively active versions of activin type I receptor (ActR-I), ActR-IB, BMPR-IA, and TGF-
type I receptor induced
(SBE)4 mediated transcription in HepG2 cells (data not
shown). Taken together these results indicate that the
(SBE)4 can be activated by different members of the TGF-
superfamily.
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DISCUSSION |
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TGF- is the prototype of a family of signaling molecules that
exhibit pleiotropic effects on cell proliferation, differentiation, as
well as on pattern formation during early vertebrate development (33).
The recent discovery of Smad proteins as downstream effectors of
signaling by activated receptors of members of the TGF-
superfamily has opened new ways for investigating regulation of target gene expression. Taking advantage of the capacity of Smad proteins to act as
ligand-independent activators of gene expression when transiently
overexpressed (24, 28), we have identified short sequences in the mouse
JunB gene promoter termed Smad binding elements (SBEs) that
mediate responsiveness to activation of intracellular signal
transduction by several members of the TGF-
superfamily. We have
shown that two SBEs in the JunB promoter form an inverted hexameric CAGACA repeat that readily binds a Smad3- and
Smad4-containing complex from TGF-
-treated Mv1Lu cells.
Bacterial-expressed full-length Smad4 and C-terminal truncated Smad3 or
Smad4 bind to the JunB SBE repeat, demonstrating that the
identified CAGACA sequence is a direct binding site for Smads. Binding
of Smad proteins is independent of the relative orientation of the SBEs
in a repeat, but multimerization of SBEs strongly increases the
affinity for Smad proteins, indicating that cooperative binding is
required for Smad function. This is demonstrated by the fact that
activation of SBE-containing reporter constructs by TGF-
or
overexpressed Smad proteins is only observed when four CAGACA elements
are present.
The JunB gene is a target for BMP2, activin, and
TGF--signaling (5-8). Ligand-mediated activation of the endogenous
promoter can be mimicked by cotransfection of a reporter construct with the pathway-restricted Smad1, Smad2, and Smad3 along with Smad4. The
CAGACA repeat isolated from the JunB gene, as present in the pGL3ti-(SBE)4 reporter construct, is also activated by
TGF-
, activin, BMP2, OP-1/BMP7, and GDF5, implicating that these
ligands activate the JunB promoter through the same response
element. Members of the BMP-subfamily have been reported to signal
through Smad1 and Smad5, while activin and TGF-
-signaling is
mediated by Smad2 and Smad3 (15). Remarkably, the SBE repeat is only efficiently bound by bacterially produced Smad3 and Smad4. Possibly, Smads 1, 2, and 5 require additional proteins for high affinity binding
to the SBE. Alternatively, and in contrast to Smad3, these Smad
proteins may associate with their target sequence only through Smad4.
Treatment of cells with TGF-
induces the phosphorylation of both
Smad2 and Smad3, which share a high degree of homology (24). However,
we could not detect Smad2 protein in the TGF-
-induced complex formed
with the 22-bp probe. The major difference between Smad2 and Smad3
resides in the DNA-binding MH1 domain. Possibly, the insertion in this
domain of Smad2 alters its binding characteristics.
The response element we have isolated from the JunB promoter
shows no homology with Mad binding site in the Drosophila
decapentaplegic-responsive vestigial quadrant enhancer has a GC-rich
core and shows no homology with the Smad binding sequence as derived
from the JunB promoter (26). Interestingly, like Smad3, Mad
binds its recognition sequence only as a C-terminal truncated protein.
These properties may be related to a proposed working mechanism for
receptor-activated Smads, in which Smads become signaling-competent
only when phosphorylation relieves the inhibitory action of their
C-terminal MH2 domain. The sequence of the JunB SBE binding
sequence shows no homology to Sp1-binding sequences that previously
have been implicated in TGF--induced transactivation of the p15 and
p21 promoters (9, 10). Smad2 and Smad4 interact with FAST-1 to form
ARF, which binds through FAST-1 to the ARE in the X. laevis
Mix.2 promoter (23). The JunB promoter nor the
pGL3ti-(SBE)4 reporter are transactivated by FAST-1 in
HepG2 cells, in contrast to a construct containing a multimerized ARE
(19).2 The ARE has been
reported to contain two 6-bp sequences that are both required for
FAST-1 binding and ARF formation. These sequences do not resemble the
JunB SBE. However, one of the 6-bp sequences overlaps with a
perfect SBE sequence. It will be of interest to investigate whether
this SBE sequence binds either Smad2 or Smad4 present in ARF. Recently,
a Smad binding site was identified in the TGF-
-responsive promoter
of the p3TP-Lux construct (25). This site contains a monomeric CAGACA
sequence identical to that of the JunB SBE and overlaps with
an AP-1 site. The CAGACA sequence appeared to be dispensable for
TGF-
induction because mutation of this sequence did not affect
TGF-
responsiveness. However, the mutated construct used in this
experiment still contained a CgGACA sequence that may be a binding site
for Smad proteins. By contrast, mutation of the AP-1 site completely
abrogated TGF-
induction. Possibly, in the context of this promoter,
Smad proteins cooperate with AP-1 to mediate TGF-
induction. In this
respect, it is noteworthy that the TGF-
-inducible PAI-1 promoter
contains three CAGACA elements that were able to mediate TGF-
, but
not BMP-responsiveness,3 of
which one element is located adjacent to an AP-1 site. Likewise, the
human
2(I) collagen promoter also harbors an AP-1 site in close
proximity to a monomeric CAGACA sequence (34). Finally, Smad proteins
may regulate gene transcription through binding to AP-1 or other
transcription factors without interacting with DNA in a manner
analogous to AP-1/steroid hormone receptor associations. The findings
may hint to a mechanism for Smad-mediated gene activation in which
receptor-activated Smad proteins cooperate with ubiquitous transcription factors. Nuclear extracts from untreated and
TGF-
-treated cells contain a constitutive binding activity that
complexes with the 22-bp inverted CAGACA repeat-containing probe. The
constitutive complex cannot be supershifted with anti-Smad antibodies.
Although we cannot rule out the possibility that the constitutive
activity corresponds with a Smad-related protein, in untreated cells
the SBE may contain a novel factor that is pre-bound to DNA to which nuclear translocated Smads bind to form a transactivating complex. We
are presently studying the JunB Smad-responsive region by
genomic footprinting to resolve this issue. This complex organization may explain our inability to show TGF-
-induction of our transiently and stably transfected JunB reporter constructs. Imitation
of the exact chromosomal context of the endogenous JunB gene
may be required for investigation of regulation of this gene by members of the TGF-
superfamily. Nevertheless, the identification of the SBE
as a binding sequence for Smad proteins now opens the way to
investigate the binding determinants of Smad proteins by site-directed
mutagenesis and elucidation of the crystal structure of the Smad/DNA
complex. Furthermore, the interactions of Smad proteins with other
proteins jointly forming a transcriptionally active complex in response
to members of the TGF-
-superfamily can now be determined.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. D. Melton for Smad1 and Smad2 cDNAs; R. Derynck for Smad3, GST-Smad3, and GST-Smad4 cDNAs; R. Lechleider for GST-Smad1 cDNA; M. Schutte for Smad4 cDNA; D. Eick for HaCaT cells; P. van der Saag for Mv1Lu and P19EC cells; and P. Coffer for NIH-3T3 cells. Activin-A was generously provided by Dr. Y. Eto (Ajinomoto Co., Inc.) and OP-1, BMP-2, and GDF-5 were generously provided by Dr. T. K. Sampath (Creative Biomolecules, Inc.). We thank Loes Drenth-Diephuis and Susanne Grimsby for technical assistance and Jean-Michel Gauthier for stimulating discussions.
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FOOTNOTES |
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* This work was sponsored by grants from the European Unity (BioMed Program BMH-CT95-0995) and the Dutch Cancer Society (KWF92-84) (to L. J., and W. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ004891.
§ Both authors contributioned equally to this work.
To whom correspondence should be addressed. Tel.:
31-50-363-2092; Fax: 31-50-363-2348; E-mail:
w.kruijer{at}biol.rug.nl.
The abbreviations used are:
AP-1, activating
protein-1; ActR, activin receptor; ARE, activin response element; BMP, bone morphogenetic protein; BMPR, BMP receptor; Erk, extracellular
signal-regulated kinase; GDF5, growth/differentiation factor-5; GST, glutathione S-transferaseMad, mother against
decapentaplegicMH, Mad homologyOP-1, osteogenic protein-1PAI-1, plasminogen activator inhibitor-1SBE, Smad binding elementTGF-, transforming growth factor-
T
R, TGF-
receptorTRE, TPA
response elementARF, activin response factorPCR, polymerase chain
reactionWT, wild typeDMEM, Dulbecco's modified Eagle's mediumTBE, Tris borate/EDTAnt, nucleotide(s)bp, base pair(s).
2 L. J. C. Jonk and W. Kruijer, unpublished observations.
3 Dennler, S., Itoh, S., Vivien, D., ten Dijke, P., Huet, S., and Gauthier, J. M. (1998) EMBO J. 17, 3091-3100.
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