Cross-talk between 1,25-Dihydroxyvitamin D3 and
Transforming Growth Factor-
Signaling Requires Binding of VDR and
Smad3 Proteins to Their Cognate DNA Recognition Elements*
Nanthakumar
Subramaniam
,
Gary M.
Leong,
Terrie-Anne
Cock,
Judith
L.
Flanagan,
Colette
Fong,
John A.
Eisman, and
Alexander P.
Kouzmenko
From the Bone and Mineral Research Program, The Garvan Institute of
Medical Research, Sydney, New South Wales 2010, Australia
Received for publication, December 7, 2000, and in revised form, January 29, 2001
 |
ABSTRACT |
1,25-Dihydroxyvitamin D3
(vitamin D) and transforming growth factor-
(TGF-
)
regulate diverse biological processes including cell proliferation and
differentiation through modulation of the expression of target genes.
Members of the Smad family of proteins function as effectors of TGF-
signaling pathways whereas the vitamin D receptor (VDR) confers vitamin
D signaling. We investigated the molecular mechanisms by which
TGF-
and vitamin D signaling pathways interact in the regulation of
the human osteocalcin promoter. Synergistic activation of the
osteocalcin gene promoter by TGF-
and vitamin D was observed in
transient transfection experiments. However, in contrast to a previous
report by Yanagisawa, J., Yanagi, Y., Masuhiro, Y., Suzawa, M.,
Watanabe, M., Kashiwagi, K., Toriyabe, T., Kawabata, M., Miyazono, K.,
and Kato, S. (1999) Science, 283, 1317-1321, synergistic
activation was not detectable when the osteocalcin vitamin D response
element (VDRE) alone was linked to a heterologous promoter. Inclusion
of the Smad binding elements (SBEs) with the VDRE in the heterologous
promoter restored synergistic activation. Furthermore, this synergy was
dependent on the spacing between VDRE and SBEs. The Smad3-Smad4
heterodimer was found to bind in gel shift assay to two distinct DNA
segments of the osteocalcin promoter:
1030 to
989 (SBE3) and
418
to
349 (SBE1). Deletion of SBE1, which is proximal to the VDRE, but
not the distal SBE3 in this promoter reporter abolished TGF-
responsiveness and eliminated synergistic co-activation with vitamin
D. Thus the molecular mechanism, whereby Smad3 and VDR mediate
cross-talk between the TGF-
and vitamin D signaling pathways,
requires both a VDRE and a SBE located in close proximity to the target promoter.
 |
INTRODUCTION |
The biological actions of 1,25-dihydroxyvitamin D3
(vitamin D)1 are mediated
through the vitamin D receptor (VDR), a member of the nuclear receptor
superfamily (1). Although VDR homodimers may play some role, most VDR
actions are thought to be mediated via VDR heterodimerization with the
retinoid X receptor (RXR) (2). The VDR/RXR heterodimers bind to vitamin
D response elements (VDREs) in the promoters of vitamin D target genes
(3, 4). VDREs typically consist of at least two copies of the consensus motif, PuG(G/T)TCA, arranged as direct repeats with a three-nucleotide spacing (DR3) (5). In addition to spacing, small differences in the
half-site sequence, and the sequence of the flanking extension of the
response elements also appear to be important in determining receptor
binding efficiency (Ref. 6 and references therein). Upon binding,
these receptors can either stimulate or repress transcription of target
genes (7), assisted by direct and indirect interaction with other
transcriptional regulatory proteins. Such additional factors may be
recruited by other regulatory signals, such as TGF-
, which has been
shown to act in synergy with vitamin D (8, 9).
Transforming growth factor-
(TGF-
), like activin and bone
morphogenetic protein (BMP), signals through the Smad family of intracellular transducing proteins (10). Among the Smads, Smad2 and
Smad3 are known to permit signaling by TGF-
and activin ligands (11). Once phosphorylated, these Smad proteins heterodimerize with
Smad4, the common mediator of TGF-
pathways. These complexes translocate to the nucleus where they regulate target genes either by
interacting with DNA or with other transcription factors (11, 12).
Though a palindromic Smad binding element (SBE), GTCTAGAC, was shown to
interact with the Smad3 and Smad4 complex, the smaller 4-bp half-site
of this SBE DNA sequence, GTCT or AGAC, has been shown to be sufficient
to bind these proteins (12, 13).
Although Smads can bind to DNA, Smad-DNA binding alone is not always
sufficient for gene activation. There are now a growing number of
examples in which Smads cooperate with DNA-binding partners to regulate
transcription (14). This type of interaction has been demonstrated for
the Mix.2, goosecoid and Xvent2 promoters where
Smads interact with FAST-1, FAST-2, mixer, milk and OAZ transcription
factors (12). DNA binding by Smad3, in conjunction with TFE3, PEBP2,
and ATF-2 to their cognate sites, has been reported to be essential for
TGF-
-dependent activation of the PAI-1,
IgA, and collagenase A promoters respectively (12). Some of
these cooperativities may be involved in mediating signals for
other signaling pathways, as suggested by recent studies that described a physical interaction of liganded VDR, Smad3, and SRC-1/TIF2 mediating
cross-talk between vitamin D and TGF-
signaling pathways (8,
9).
In the present study, we identified functional TGF-
-response
elements in the human osteocalcin (hOC) gene promoter;
demonstrated cross-talk between vitamin D and TGF-
signaling
pathways in regulation of this promoter; and determined the molecular
mechanism for synergistic activation of the hOC promoter by
vitamin D and TGF-
.
 |
EXPERIMENTAL PROCEDURES |
Plasmid Constructs--
Reporter plasmids pOSLUC2 (
1.3 kb) and
pOSLUC1 (
344 bp), of the human osteocalcin gene promoter linked
upstream of the luciferase reporter of pGL-3basic have been described
previously (15). pOSLUC3 (
1.3 kb to
1111 plus
905 to
833) was
created by deletion of a StuI fragment from pOSLUC2.
Deletion of the PstI and SacI fragment from
pOSLUC2 resulted in pOSLUC4 (
1.3 kb to
833 plus
341). pOSLUC5
(
1.3 kb to
341 plus
191) was constructed by deletion of the
SacI and ApaI fragment from pOSLUC2. pOSLUC6 was created by cloning in an osteocalcin promoter PCR fragment (forward primer
1337 to
1311 with MluI 5' overhang and reverse
primer
475 to
500 with XhoI 5' overhang) into the pGL-3
promoter vector. The p3TP-Lux reporter, which contains SBEs from the
plasminogen activator inhibitor-1 (PAI-1) gene in front of
the luciferase reporter, was a kind gift from Dr. J. Massague (16).
Double-stranded oligonucleotides corresponding to human osteocalcin
VDRE (
507 to
470 or
515 to
484) or rat osteocalcin VDRE (
515
to
484) were cloned upstream of the SV40 promoter of pGL-3 promoter
vector to produce hVDREluc and rVDREluc respectively. hVDRE-3TPlux was created by inserting the hVDREs in front of the SBEs from the p3TP-Lux
plasmid. Insertion of 450 bp fragment, which do not contain known SBEs
from pSG5 expression vector, between VDRE and SBEs of hVDRE-3TPlux
into XhoI site resulted in the V450T reporter plasmid. VDR
expression vector, mVDR-pSG5 and FLAG-Smad2,3,4/pcDNA3 plasmids
that express Smad2, 3, or 4 with N-terminal FLAG tags have been
described elsewhere (15, 17). FLAG-S3-(41-435) that expresses Smad3
with an N-terminal 41 amino acid deletion was a kind gift from Dr S. Kato (8).
Cell Culture and Transfections--
COS-1 cells were grown in
Dulbecco's modified Eagle medium supplemented with 5% fetal calf
serum, 2 mM L-glutamine, 100 IU/ml penicillin
and 100 µg/ml streptomycin (all from Life Technologies, Inc.) at
37 °C in 5% CO2. Cells were plated at ~30%
confluence in 24-well plates and the following day were transfected
using FuGENE-6 transfection reagent (Roche Molecular Biochemicals,
Indianapolis, IN) with 250 ng/well of reporter plasmid and 50 ng of the
VDR or Smad expression plasmid. After a 6-h incubation at 37 °C in 5% CO2, 2% charcoal stripped fetal calf serum containing
medium was added having either 10 nM 1,25-dihydroxy vitamin
D3 (Hoffman-LaRoche) or vehicle (isopropyl alcohol, final
conc. 0.01%) and/or TGF-
(1 ng/ml) (Sigma, Saint Louis, MI) or
vehicle (4 nM HCl in 1 mg/ml bovine serum albumin).
Following incubation for 16 h, cells were lysed with 2× Promega
lysis buffer, and luciferase assays were performed in triplicate with
the firefly luciferase assay kit (Promega, Madison, WI) and measured
using a Berthold LB953 Autolumat luminometer (Berthold, Bad Wildbad,
Germany). Each experimental condition was measured in triplicates, and
the values given represent the mean ± S.D. from two to three experiments.
Gel Shift Assays--
Oligonucleotides were endlabeled by T4
polynucleotide kinase using [
-32P]ATP (3,000 Ci/mmol,
Amersham Pharmacia Biotech). DNA binding reactions of 20 µl were
carried out in buffer containing 20 mM Tris-Cl, pH 8.0, 10% (W/V) glycerol, 1 mM EDTA, 1 mM
dithiothreitol, 400 ng of poly(dI-dC) (Amersham Pharmacia Biotech), 75 mM KCl, 3% bovine serum albumin, 0.1-0.3 ng of
radiolabeled oligonucleotide, and 2 µl of Smad3 and Smad4 proteins
transcribed and translated in vitro, in rabbit reticulocyte
lysate (Promega, Madison, WI). Binding reactions were performed in room
temperature for 20 min. Free and bound DNA were separated on 4%
polyacrylamide (acrylamide/bisacrylamide, 29:0.5) gels, which were run
at a constant voltage of 250 V in 22 mM Tris-borate, 0.5 mM EDTA (6).
 |
RESULTS |
Vitamin D and TGF-
Synergistically Activated Human
Osteocalcin Promoter Activity--
The modulatory effects of
1,25-dihydroxyvitamin D3 and TGF-
signaling on the human
osteocalcin promoter were analyzed by transient transfections into COS1
cells using the pOSLUC2 reporter and VDR expression plasmid.
1,25-Dihydroxyvitamin D3 transactivated the pOSLUC2
reporter activity by 6-fold whereas TGF-
alone augmented the
reporter activity 2-fold (Fig. 1). Both
ligands, produced a synergistic effect (10-fold induction). This
synergy was specific to the pOSLUC2 reporter, because these two ligands
together or singly did not significantly alter transcriptional activity
of pOSLUC1, which lacks 960 bp of distal 5' DNA sequence from the human
osteocalcin promoter including the VDRE (15). Smad3 and Smad2, but not
BMP Smads (Smad1 or Smad5), activated the osteocalcin promoter reporter
(data not shown), both in the absence and presence of TGF-
.
Augmentation of reporter activity was greater with Smad3, suggesting a
putative Smad3 binding site conferred the TGF-
signal for this
promoter. Overexpression of Smads 3 and 4 constitutively activated
basal promoter activity mimicking TGF-
signaling, as has been
reported for other well characterized TGF-
responsive promoters (11,
12).

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Fig. 1.
1,25-dihydroxyvitamin D3
(VD3) and
TGF- -activated OC
promoter. The human osteocalcin promoter conferred both
1,25-dihydroxyvitamin D3 and TGF- signaling to a
luciferase reporter. The pOSLUC2 or pOSLUC1 reporter was transiently
co-transfected with VDR expression plasmid into COS1 cells, and
1,25-dihydroxyvitamin D3 (VD3) and/or
TGF- -induced luciferase activity was measured and related to the
uninduced reporter activity.
|
|
TGF-
Did Not Augment 1,25-Dihydroxyvitamin D3
Activation of VDRE-dependent Reporters--
To
investigate whether the synergistic activity observed in our previous
experiment was in fact mediated through the VDRE (4), the hVDREluc
reporter was co-transfected with a VDR expression plasmid into COS1
cells. Whereas 1,25-dihydroxyvitamin D3 increased the
reporter activity 10-fold, TGF-
did not affect reporter activity (Fig. 2A), nor was there any
synergy between 1,25-dihydroxyvitamin D3 and TGF-
, as
observed in the case of the pOSLUC2 reporter. As the DR3 VDRE in the
human osteocalcin promoter overlaps a DR6 VDRE and an AP1 site, testing
of these overlapping elements (
515 to
484) was carried out in a
luciferase reporter construct. This construct also responded to
1,25-dihydroxyvitamin D3 but did not respond to TGF-
,
and there was no synergy between 1,25-dihydroxyvitamin D3
and TGF-
(Fig. 2B). Comparable experiments performed with the rVDREluc reporter had a similar 1,25-dihydroxyvitamin
D3 but no response to TGF-
and no synergy between
1,25-dihydroxyvitamin D3 and TGF-
signals, irrespective
of nucleotide differences in human and rat VDREs (Fig. 2C).
These results suggest that VDRE alone is not sufficient to mediate the
synergy.

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Fig. 2.
VDRE-linked heterologous promoters were not
sufficient to convey TGF- signaling to a
luciferase reporter. hVDREluc (DR3, A),
hVDREluc (DR3+6, B), and rVDREluc (C)
reporters were transiently co-transfected with VDR expression plasmid
into COS1 cells, and 1,25-dihydroxyvitamin D3
(VD3) and/or TGF- -induced luciferase activity was
measured and related to each uninduced reporter activity.
|
|
1,25-Dihydroxyvitamin D3 Did Not Augment
TGF-
Induction of a TRE-dependent
Reporter--
Possible effects of 1,25-dihydroxyvitamin D3
on TGF-
transcriptional activation were tested on a heterologous
reporter construct carrying the well characterized TRE from the
PAI-1 gene (p3TP-Lux) in COS1 cells (16). Whereas TGF-
activated the reporter in these cells 7-fold, 1,25-dihydroxyvitamin
D3 had no effect on the basal activity of the reporter
(Fig. 3A). Moreover,
TGF-
-activated transcription was not augmented by
1,25-dihydroxyvitamin D3 (Fig. 3A). This result
suggests that the TRE alone is incapable of converging vitamin D and
TGF-
signaling pathways.

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Fig. 3.
Presence of a TRE sequence was essential to
convey TGF- signaling through VDRE-linked
heterologous promoters. p3TP-Lux (A) and hVDRE-3Tplux
(B) reporters were transiently co-transfected with VDR
expression vector into COS1 cells, and 1,25-dihydroxyvitamin
D3 (VD3) and/or TGF- -induced luciferase
activity were measured in luciferase units. All luciferase activities
were related to uninduced reporter activity.
|
|
VDRE Coupled to the TRE Sequence Restored Synergy by Vitamin D and
TGF-
Signaling Pathways--
To explore whether the synergy by
1,25-dihydroxyvitamin D3 and TGF-
pathways required
concomitant binding of both VDR and Smad to their separate cognate
elements, the hVDRE-3TPlux reporter plasmid, which contains both
respective response elements, was co-transfected with VDR expression
plasmid in COS1 cells. 1,25-Dihydroxyvitamin D3 or TGF-
alone activated this reporter approximately by 10-fold (Fig.
3B). Importantly, hVDRE-3TPlux reporter responded
synergistically to 1,25-dihydroxyvitamin D3 and TGF-
(45-fold), suggesting both binding sites are needed to mediate the
synergy (Fig. 3B).
Smad3-Smad4 Heterodimer Binding to Osteocalcin
Promoter--
Sequence analysis of the human osteocalcin promoter
identified three regions:
415 to
353 (SBE1);
313 to
228 (SBE2);
and
1027 to
997 (SBE3); each of which contains three copies of GTCT or its reverse complement AGAC, an optimal target/binding site for
Smad3 and Smad4 (13). Gel shift assays using Smad3 and Smad4 proteins,
transcribed and translated in vitro in rabbit reticulocyte lysate, were used to examine DNA binding activity of these putative SBEs as well as the PE2 fragment of the PAI-1 promoter (13) as a positive Smad binding control. SBE1 and SBE3 formed complexes similar to PE2 (Fig. 4) whereas there was
no complex formation with SBE2 (data not shown). Rabbit reticulocyte
lysate alone did not form any complexes with these DNA probes (data not
shown). DNA binding specificity of complexes formed with DNA probes,
was examined using cold DNA probes as competitors. Both SBE1 and SBE3 inhibited complex formation. However the PE2 element (
586 to
551)
from the PAI-1 promoter, which contained three AGAC motifs, moderately inhibited the complex formation with either SBE1 or SBE3.

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Fig. 4.
Smad3 and Smad4 binding to SBE1 and SBE3
in vitro. In vitro-produced Smad3 and
Smad4 proteins (2 µl) were incubated with radiolabeled SBE1, SBE2,
SBE3, and PE2 and analyzed by gel shift assay. A 50-fold excess of
unlabeled SBE1, SBE3, or PE2, a fragment of the PAI-1
promoter (13), were included in the incubation reaction before the
assay.
|
|
Smad3-Smad4-mediated TGF-
-responsive Regions in the Osteocalcin
Promoter--
To determine functional significance of the Smad binding
to the potential SBEs in the promoter, we made deletions of larger regions from the pOSLUC2 (
1.3 kb) that removed SBE2, SBE1, and/or SBE3 (pOSLUC3-6). Cotransfection of Smad3 and Smad4 expression vectors with reporter plasmids pOSLUC1-6 augmented basal and
TGF-
-dependent activity (Fig.
5). The highest activation was
observed with the wild-type promoter reporter, pOSLUC2. Deletion of
SBE1 (pOSLUC4) greatly affected Smad3-mediated augmentation whereas a
moderate reduction was observed with the deletion of SBE3 (pOSLUC3),
both of which reflected the previous binding studies that SBE1 and SBE3
were capable of binding to Smad3 and Smad4. Deletion of SBE2 (pOSLUC5),
which did not bind with Smad3 and Smad4 in the gel shift assay (data
not shown), did not affect the reporter activity. Combined deletion of
SBE1 and SBE3 (pOSLUC1) or SBE1 and SBE2 (pOSLUC6) also resulted in
marked loss of reporter activity. These results suggest that the
Smad-mediated activity was largely through the SBE1.

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Fig. 5.
Smad3 and Smad4 activated the osteocalcin
promoter mainly through SBE1. pOSLUC1, pOSLUC2, pOSLUC3, pOSLUC4,
pOSLUC5, and pOSLUC6 reporters were co-transfected with empty vector or
Smad3 and Smad4 expression plasmids into COS-1 cells, and cells were
treated with or without TGF- . All luciferase activities were related
to individual uninduced reporter activity.
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|
VDRE and TRE Spacing Crucial for 1,25-Dihydroxyvitamin
D3 and TGF-
Synergy--
The role of the
spacing between VDRE and TRE in the 1,25-dihydroxyvitamin
D3 and TGF-
synergy was examined using V450T reporter plasmid. This construct, containing both VDRE and TRE separated by 450 bp, was co-transfected with VDR expression plasmid in COS1 cells. This
reporter activation by 1,25-dihydroxyvitamin D3 or TGF-
alone or in combination was remarkably reduced (Fig.
6) in comparision to the parent
hVDRE-3TPlux reporter plasmid (Fig. 3B). The level of
reduced synergy conferred by V450T was comparable with pOSLUC6 reporter
that has the VDRE and SBE3 at a similar 450-bp distance. This result
demonstrated that synergy by 1,25-dihydroxyvitamin D3 and
TGF-
signaling, conferred via their respective transducers VDR and
Smad3, only occurred through binding to VDRE and TRE located in the
close proximity in the target promoter.

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Fig. 6.
Spacing between VDRE and TRE affects
synergy. The V620T, V450T, or pOSLUC6 reporters were transiently
co-transfected with VDR expression plasmid into COS1 cells, and
1,25-dihydroxyvitamin D3 (VD3) and/or
TGF- -induced luciferase activity were measured and related to
individual uninduced reporter activity.
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Direct Interaction between VDR and Smad3 Also Required for
Synergy--
It was previously demonstrated that the 40-amino acid
N-terminally truncated Smad3 did not physically interact with VDR in either GST pull-down assays or the mammalian two-hybrid system, whereas
the 20-amino acid N-terminally truncated Smad3 retained such abilities
(8). We examined whether these Smad3 mutants together with VDR were
capable of synergistically activating the hVDRE-3TPlux reporter. The
wild-type Smad3 and Smad3-(21-435), capable of transducing TGF-
signal, together with VDR synergistically transactivated this reporter
(Fig. 7). Smad3-(41-345) failed to mediate TGF-
signaling, and with VDR, was unable to transactivate synergistically as Smad3, suggesting that a competent Smad3 in addition
to separate and distinct binding sites is also required for the
synergy.

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Fig. 7.
Competent Smad3 required for synergy in
vivo. hVDRE-3TPlux reporter was transiently
co-transfected with VDR and in the absence or presence of Smad3 or
S3-(21-435), S3-(41-435) expression vector into COS-1 cells, and 1, 25-dihydroxyvitamin D3 (VD3) and/or
TGF- -induced luciferase activity was measured and related to
uninduced reporter activity.
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 |
DISCUSSION |
DNA-binding nuclear factors interact with one another and with
cofactors to form complex networks of DNA-protein-protein interactions for transcriptional regulation in eukaryotic cells (5, 8). Transcription regulatory regions of eukaryotic gene promoters generally
consist of clusters of transcription factor-binding sites that
coordinate responses to combinations of signaling molecules and
different transcription factors utilize a variety of means to promote
or repress target genes. (19). Osteocalcin gene expression is
facilitated by overlapping and contiguous regulatory elements including
a functional VDRE in the distal promoter region (4, 20, 21). Regulatory
factors, including VDR, Cbfa-1, AP-1 factors, and homeodomain
proteins act directly, and BMPs and TGF-
act indirectly to mediate
commitment to the osteoblastic cell lineage and regulation of the
osteocalcin promoter (22, 23).
Only two reports to date describe transcriptional regulation through
protein-protein interaction involving Smad3 and nuclear hormone
receptors (8, 24). Whereas Yanagisawa et al. (8) reported
that activation by VDR, but not other receptors (oestrogen receptor
, androgen receptor, GR, retinoic acid receptor, and RXR), was
affected by the treatment with TGF-
, Song et al. (24) found that Smad3 was the direct target for repression by the GR on a
type-1 PAI-1 gene promoter. They provided evidence for
GR-Smad3 interaction in vivo and in vitro,
although they could not rule out the possibility that other proteins
might have mediated the interaction.
A structure/function analysis of the hOC promoter was
initiated to explore its synergistic activation by vitamin D and
TGF-
(Fig. 1). Recently it has been reported that Smad3, a
downstream component of the TGF-
signaling pathway, acts as a
coactivator of VDR potentiating its ligand-induced transactivation (8, 9). Hence, we tested the VDRE from the hOC promoter as a
separate response element in the context of a minimal heterologous
promoter. There was no synergy between 1,25-dihydroxyvitamin
D3 and TGF-
signals through this VDRE (Fig.
2A). In addition, the slightly longer VDRE, which contained
both DR6 and DR3 spacing, exhibited no synergy in transient
transfection studies in COS-1 cells by these signals (Fig.
2B). A rat osteocalcin DR3-type of VDRE placed in
heterologous context also did not mediate synergistic effects of
1,25-dihydroxyvitamin D3 and TGF-
(Fig. 2C).
These results suggested that nucleotide differences in different
minimal VDREs cannot explain the failure to observe TGF-
-mediated
reporter activity. In addition, similar results were obtained in three separate cell lineages, suggesting differences in cellular context could not be responsible for the disparity of the results. These data
are not consistent with direct protein-protein interaction as the sole
mode of action to explain the synergy, and therefore are in sharp
contrast to the findings of Yanagisawa et al.(8, 9) who
reported that the VDRE alone was sufficient for convergence of
1,25-dihydroxyvitamin D3 and TGF-
signaling pathways.
Disparity between our results and previously reported results may be
attributed to the design of the reporter construct used in the
transient transfection studies. Constructs in Ref. 8 derived from pGCAT
(25) and contained a synthetic oligonucleotide insert with
HindIII and XbaI sites. This XbaI site
follows GT nucleotide of the parental plasmid sequence and creates a
DNA sequence 5'-GTCT-3', which has been defined as a Smad box (12) and
could form part of a cryptic SBE in close proximity to the VDRE.
Potentially, the presence of these two closely spaced sites could be
mediating the synergy between 1,25-dihydroxyvitamin D3 and
TGF-
signals in their reporter construct. Irrespective of these
reasons, in our studies, convergence of vitamin D and TGF-
signaling
required both specific DNA response elements in both heterologous
promoter model constructs and in the human osteocalcin promoter.
The ability of the Smad3-Smad4 complex alone or in the presence of
TGF-
to transactivate the OSLUC2 reporter is consistent with the
existence of Smad binding sites in this promoter. This led us to test
the activation of reporters containing both a VDRE (human) and a
cluster of TGF-
response elements from the PAI-1 gene,
upstream of the SV40 promoter. Transient transfection studies with
these reporter constructs recreated the synergy between vitamin D and
TGF-
signals, with activation by vitamin D comparable with the
induction of hVDREluc reporter (Fig. 2A), and TGF-
stimulated activation comparable with that of the standard TGF-
responsive reporter p3TP-Lux (Fig. 3A). Of three potential
SBE clusters in the osteocalcin promoter, two (SBE1 and SBE3) bound to
Smad3 and Smad4 proteins in a gel shift assay (Fig. 4). Deletion of
these two putative regions separately or together reduced
TGF-
-mediated reporter activity confirming their functional role
(Fig. 5). Taken together, these results suggest that the convergence of
TGF-
and vitamin D signaling does not take place in the absence of either VDRE or TRE. In agreement with these data, a Smad3-Smad4 complex
and an AP-1 complex have been reported to synergize in transcriptional
activation of the c-jun promoter by binding to separate
sites 120 bp apart (26). Similarly, transcriptional cooperativity
between c-Jun and Smad3 at the AP-1 binding site of the collagenase I
promoter also required the interaction of c-Jun and Smad3 with DNA
(27). Several other recent reports point to cross-talk between Smad
proteins and transcription factors such as TFE3, FAST-1, SP1, CREB, and
Tinman in the expression of various TGF-
-inducible genes (Ref. 13
and references therein). It has been proposed that the number of SBEs
as well as discrete spacer segments between SBEs and/or Smad-partner
transcription factor binding site(s) will also be critical for the
specificity of TGF-
-induced transcription of these promoters
(13). Our study also found that the synergy by vitamin D and TGF-
was reduced when the spacing between VDRE and TRE was extended (Fig.
6).
The promoter studies further indicate that simultaneous binding of VDR
and Smad3 to their cognate sequences (Fig. 3B) and possibly
a direct interaction between these two proteins (Fig. 7), are both
required for the convergence of the vitamin D and TGF-
signals. The
Smad3-(41-345) construct failed to mediate TGF-
signaling through
the 3TPlux reporter (data not shown) suggesting that this Smad3 mutant
is functionally inert. The same N-terminal deletion mutant failed to
interact with VDR (8). Our study also found that this mutant was unable
to transactivate synergistically with VDR through hVDRE-3TPlux
reporter, therefore we could not positively conclude that a direct
VDR-Smad3 interaction was involved in the cross-talk between these
regulatory pathways.
In the present study we demonstrated that the VDRE-containing
hOC gene promoter contains two functional Smad3 binding
elements through which the TGF-
and vitamin D signaling pathways
converge. Our data thus strongly suggest that cross-talk between these
two signaling pathways requires the presence in the promoter of both respective response elements in close proximity. These data support the
concept of specific response elements being required to transduce both
single agents and to integrate the synergistic activation of hormone
responsive genes.
 |
ACKNOWLEDGEMENTS |
p3TP-Lux luciferase reporter plasmid was a
kind gift from Dr. J. Massague, and FLAG-Smad2,3,4/pcDNA3
plasmids that express Smad2,3 or 4 with N-terminal FLAG tags have been
kindly provided by Drs. K. Miyazono and T. Imamura. FLAG-S3-(41-435)
was a kind gift from Dr. S. Kato. We also thank Dr. S. Kato for
encouragement and valuable discussions and Drs. E. M. Gardiner, G. Thomas, R. Daly, and M. Henderson for valuable comments on the manuscript.
 |
FOOTNOTES |
*
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.
To whom correspondence should be addressed: Muscle Development
Unit, Children's Medical Research Institute, Wentworthville NSW 2145, Australia. Tel.: 61-2-9687-2800l; Fax: 61-2-9687-2120; E-mail:
nsubramaniam@cmri.usyd.edu.au.
Published, JBC Papers in Press, February 5, 2001 DOI 10.1074/jbc.M011033200
 |
ABBREVIATIONS |
The abbreviations used are:
vitamin D or
VD3, 1,25-dihydroxyvitamin D3;
VDR, vitamin D receptor;
RXR, retinoid X receptor;
VDRE, vitamin D response element;
DR, direct
repeat;
TGF-
, transforming growth factor-
;
BMP, bone
morphogenetic protein;
SBE, Smad binding element;
luc, luciferase;
GR, glucocorticoid receptor;
hOC, human osteocalcin;
PAI-1, plasminogen
activator inhibitor-1;
bp, base pair;
kb, kilobase pair.
 |
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