From the University of Minnesota Cancer Center, Department of
Orthopaedic Surgery and Graduate Program in Microbiology, Immunology
and Cancer Biology, Minneapolis, Minnesota 55455
Received for publication, November 8, 2002, and in revised form, January 24, 2003
Functional control of the transcription factor
Runx2 is crucial for normal bone formation. Runx2 is detectable
throughout osteoblast development and maturation and temporally
regulates several bone-specific genes. In this study, we identified a
novel post-translational mechanism regulating
Runx2-dependent activation of the osteocalcin promoter. A
functional binding site for the high mobility group protein lymphoid
enhancer-binding factor 1 (LEF1) was found adjacent to the proximal
Runx2-binding site in the osteocalcin promoter. In transcription
assays, LEF1 repressed Runx2-induced activation of the mouse
osteocalcin 2 promoter in several osteoblast lineage cell lines.
Mutations in the LEF1-binding site increased the basal activity of the
osteocalcin promoter; however, the LEF1 recognition site in the
osteocalcin promoter was surprisingly not required for LEF1 repression.
A novel interaction between the DNA-binding domains of Runx2 and LEF1
was identified and found crucial for LEF1-mediated repression of Runx2.
LEF1 is a nuclear effector of the Wnt/LRP5/
-catenin signaling
pathway, which is also essential for osteoblast proliferation and
normal skeletal development. A constitutively active
-catenin
enhanced LEF1-dependent repression of Runx2. These data
identify a novel mechanism of regulating Runx2 activity in osteoblasts
and link Runx2 transcriptional activity to
-catenin signaling.
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INTRODUCTION |
Runx2 (Cbfa1, AML-3, PEBP2
A) is one of three mammalian Runt
domain factors and is essential for bone formation (1).
Runx2 deficiency is developmentally lethal because it
inhibits osteoblast development, chondrocyte differentiation in some
skeletal elements, and vascular invasion into cartilage to prevent
skeletal ossification (2-4). Moreover, a dominant-negative Runx2
protein prevents osteoblast differentiation in adult mice without
decreasing osteoblast numbers (5). Runx2 haploinsufficiency
hinders intramembranous bone formation and causes the rare skeletal
disorder, cleidocranial dysplasia (6). Interestingly, Runx2
overexpression induces bone fragility by blocking osteoblast
differentiation and enhancing bone resorption (7, 8). These genetic
studies demonstrate that cellular control of Runx2 expression levels
and function is crucial for skeletal development.
At the molecular level, Runx2 activity is regulated by multiple
transcriptional and post-translational mechanisms (9). Runx proteins
activate or repress gene expression by binding the DNA sequence,
TGPuGGTPu (10). Runx-binding sites are often necessary but are not
sufficient for transcriptional regulation of tissue-specific genes;
thus, it has been hypothesized that Runx proteins are organizers that
facilitate the assembly of transcriptional regulatory complexes on gene
regulatory elements (11, 12). Runx factors, in fact, interact with
numerous transcription factors (e.g. AP1, C/EBP, Ets1, and
SMADs) and transcriptional co-factors (e.g. p300, ALY, mSin3A, TLE, and HDAC6) to regulate tissue-specific gene expression (13-16). Runx1-dependent trans-activation of
the T cell receptor enhancer is also increased indirectly by
DNA-binding proteins, such as
LEF1,1 which bends DNA to
facilitate long distance cooperative interactions between Runx1 and
Ets1 (17, 18).
LEF1 is a high mobility group (HMG) protein and nuclear effector of the
canonical Wnt signaling pathway (19, 20). LEF1 binds the consensus DNA
sequence C/TCTTTGAA in the minor groove, creates a 130° bend in the
double helix, and alters of binding of other transcription factors to
neighboring sites (17, 21). LEF1 transcriptional activity is regulated
by interactions with transcriptional co-activators and co-repressors.
In the absence of Wnt signals, LEF1 binds to transcriptional
co-repressors TLE, CtBP, and HDACs to inhibit gene expression (22).
Wnts convert LEF1 into a transcriptional activator by stimulating the
disassembly of the GSK-3-Axin-APC multi-protein complex to prevent
ubiquitin-mediated degradation of
-catenin (22, 23). Increased
-catenin expression facilitates its translocation to the nucleus
where it displaces co-repressors from LEF1, binds LEF1, and recruits
co-activators to induce gene transcription (22, 23). LEF1,
c-myc, and cyclin D1 are among many genes whose
promoters are activated by the Wnt/
-catenin/LEF1 pathway (24-26).
It was recently demonstrated that this canonical pathway also directly
represses gene transcription in a manner dependent upon Wnt
concentrations. In Drosophila, Wnt/wingless (Wg) induces
Armadillo (
-catenin homologue) and dTCF1/Pangolin (LEF1 homologues)
to repress transcription of shavenbaby, stripe, decapentaplegic, and daughterless (27-30). The
mechanism of Wnt-induced transcriptional repression through
LEF1-binding sites has not yet been defined, but these studies
demonstrate that Wnts stimulate multiple, complex nuclear events.
The canonical Wnt signaling pathway regulates bone density by
regulating postnatal osteoblast proliferation (31). Inactivating mutations in Wnt receptor, LRP5, decrease bone mass accrual during growth and cause the autosomal-recessive disorder
osteoporosis-pseudoglioma syndrome in humans and mice by decreasing
osteoblast proliferation and without altering Runx2 mRNA levels
(32, 33). Conversely, activating mutations in LRP5 are
linked to autosomal-dominant high bone mass traits and increased
osteoblast proliferation (34, 35). The downstream effects of Wnt
signaling on osteoblast-specific gene transcription have not yet been
described. In this report, we demonstrate that
Runx2-dependent activation of the most osteoblast-specific gene, osteocalcin, is repressed by LEF1. A constitutively activated
-catenin protein enhances LEF1-mediated repression of Runx2. These
data suggest that in addition to stimulating osteoblast proliferation,
the Wnt signaling pathway may slow differentiation by inhibiting Runx2 activity.
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EXPERIMENTAL PROCEDURES |
Plasmids--
Mammalian expression plasmids (pCMV5) for Runx2
(MRIPV and MASNSL isoforms) were provided by Dr. Scott Heibert and Dr.
Gary Stein, respectively. pCMV5-LEF1-HA was obtained from Dr. Rudolf Grosschedl. LEF1 mutants were generated from pCMV5-LEF1-HA with gene-specific oligonucleotides (primer sequences available upon request) and PCR. PCR products were cloned into pPCR-Script Amp SK(+)
(Stratagene). GAL fusion proteins were constructed by subcloning LEF1 fragments from pPCR-Script into pENTR-3C (Invitrogen) with DraI and EcoRI and then recombining them into the
pDEST-GAL(M2) vector with the GATEWAY Clonase Enzyme mix (14). The
-catenin vector Xb/pcDNA3.1+ was provided by Dr. Jennifer Hall
(36). The osteocalcin promoter reporters mOG2-luc and p6OSE2-luc were provided by Dr. Gerard Karsenty (37). The GAL-responsive pAH205 was
provided by Dr. Vivian Bardwell. Mutant mOG2-luc reporters were
generated using QuikChange (Stratagene) and gene-specific oligonucleotides containing mutations in the Runt and LEF1-binding sites (mutations are depicted in Fig. 1A; primer sequences
are available upon request.)
Cell Lines and Transfection--
The murine preosteoblast cell
line MC3T3 (clone E1, passage 20 or less), murine mesenchymal precursor
cell line C3H10T1/2, and rat osteosarcoma cell line UMR-106 (clone
106.01) were cultured in minimum essential medium supplemented with
10% fetal bovine serum, 292 mg/liter L-glutamine, 1×
nonessential amino acids, 100 units of penicillin, and 100 µg/ml
streptomycin. Prior to transfection, MC3T3 and C3H10T1/2 were seeded in
6-well plates at a density of 1.25 × 105 cells/well.
UMR-106 cells were seeded in 12-well plates at a density of 7 × 104 cells/well. MC3T3, C3H10T1/2, and UMR-106 cells were
transiently transfected with mOG2-luc (300 ng, 6-well; 100 ng,
12-well), pRL-TK (50 ng, 6-well; 5 ng, 12-well) or pCMV-SEAP (500 ng,
6-well), and expression plasmids pCMV5-LEF1-HA, pCMV5-Runx2 (MRIPV or
MASNS isoform), and
-catenin as indicated with either LipofectAMINE or LipofectAMINE 2000 (Invitrogen) and as directed by the manufacturer. COS cells were transfected using DEAE-dextran. For differentiation assays, MC3T3 cells were seeded at a concentration of 2000 cells/cm2 and cultured in
-minimum essential medium
supplemented with 10% fetal bovine serum, 50 µg/ml ascorbic acid, 10 mM
-glycerol phosphate, 100 units/ml penicillin, and 100 µg/ml streptomycin.
Immunofluorescence--
MC3T3 cells grown on coverslips were
washed twice with PBS and fixed at room temperature for 30 min in 4%
paraformaldehyde. Fixed cells were lysed for 5 min at room temperature
in 0.3% Triton X-100 in PBS and blocked for 30 min at room temperature
in immunofluorescence solution (3% bovine serum albumin, 20 mM MgCl2, 0.3% Tween 20, PBS). The cells were
then incubated with either anti-LEF-1 (REMB1, Exalpha Biologicals) or
anti-
-catenin (BD Biosciences) diluted to 1 µg/ml in
immunofluorescence solution for 1 h at 37 °C in a humidified
chamber. Excess primary antibody was removed by washing three times for
5 min in PBS with 0.1% Triton X-100. The cells were incubated for 30 min with goat anti-mouse IgG conjugated to Alexa 546 (1:50 dilution in
immunofluorescence solution) at 37 °C in a humidified chamber.
Excess secondary antibody was removed by washing three times for 5 min in PBS with 0.1% Triton X-100. The nuclei were stained with 5 µg/ml Hoescht 3258 for 5 min. The coverslips were mounted on glass
microscope slides and viewed with fluorescence microscopy.
Transcription Assays--
The cells were harvested 2 days
post-transfection, and luciferase expression was measured using a
luciferase assay kit or a dual luciferase assay kit (Promega). Firefly
luciferase activities were normalized to either human placental
secreted alkaline phosphatase (SEAP) or Renilla luciferase.
SEAP values were determined as previously described (38).
In Vitro Protein Interaction Studies--
LEF1 proteins were
in vitro transcribed and translated from pPCR-Script-LEF1 in
the presence of 35S-labeled amino acids using rabbit
reticulocyte lysates (Promega). GST and GST-Runx fusion proteins were
produced in Escherichia coli and extracted in lysis buffer A
(10 mM MES, pH 6.5, 150 mM NaCl, 2 mM MgCl2, 0.5 mM EDTA, 0.5% Triton
X-100, 5 mM dithiothreitol, and protease inhibitors
(aprotinin, leupeptin, phenylmethylsulfonyl fluoride), and 50 µg/ml
ethidium bromide as indicated) with sonication. GST proteins were
purified with glutathione-Sepharose beads and incubated with
radiolabeled LEF1 proteins in the presence or absence of 50 µg/ml
ethidium bromide. The beads were then washed three times with buffer A,
resuspended in Laemmli buffer, boiled, and pelleted by centrifugation.
The proteins in the supernatant were resolved by SDS-8% PAGE. The gel
was fixed with 40% methanol and 10% acetic acid, incubated with
Amplify (Amersham Biosciences), dried, and exposed to film.
Electrophoretic Mobility Shift Assays--
Double-stranded
probes derived from the mOG2 osteocalcin promoter (wild type sequence:
5'-TTCAATCACCAACCACAGCATCCTTTGGGTTTGAC-3') were end-labeled with
[
-32P]dATP using Klenow DNA polymerase. COS cell
lysates (5 mg) expressing Runx2 or LEF1 (HA-tagged) were incubated with
an equal volume of 2× probe mix (100 mM HEPES, pH 7.8, 5 mM MgCl2 0.5 mM EGTA, pH 8.0, 2 mM dithiothreitol, 200 mM KCl, 8.25% Ficoll,
12.5 µg/ml salmon sperm DNA, and 41.25 pg/ml probe) at room
temperature for 30 min. For supershift assays, 240 µg/ml of
anti-Runx2 (39) or HA antiserum (Sigma) was added to the binding
reaction for 30 min on ice. Unlabeled consensus binding sites for
either Runx (5'-AATTCGAGTATTGTGGTTAATACG-3') or LEF1
(5'-AATTCCGGCCTTTGATCTTTGCTA-3') were used at a concentration of 100×
the probe concentration to compete with binding to the labeled probe.
Protein-DNA complexes were resolved by nondenaturing 4% acrylamide gel
electrophoresis and visualized by autoradiography.
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RESULTS |
LEF1 Binds the Osteocalcin Promoter--
In an effort to discover
novel mechanisms that regulate Runx2-dependent
trans-activity, we identified a sequence adjacent to the
crucial proximal Runx2-binding site on the murine osteocalcin promoter,
mOG2 (37), that resembles the consensus LEF1 recognition site (Fig.
1A). The LEF1 recognition
sequence was separated from the proximal Runx-binding site by just four
base pairs. The LEF1 binding sequence and its relative location to the
Runx site are conserved in mouse, rat, and human osteocalcin promoters.
To determine whether LEF1 binds the mOG2 promoter, electrophoretic
mobility shift assays (EMSAs) were performed with radiolabeled DNA
probes containing mOG2 residues,
144 to
110 (Fig. 1A).
COS lysates expressing LEF1-HA produced two complexes that hindered the
migration of the probe (Fig. 1B). These bands were
specifically competed with either unlabeled double-stranded
oligonucleotides containing a wild type LEF (L) consensus binding site
or HA-specific antibodies. Oligonucleotides containing a mutant
LEF-binding site (mtL) or a Runx2 antibody did not alter the LEF
complexes (Fig. 1B). Runx2 lysates produced a single band
when incubated with the mOG2 probe. This band was specifically competed
by the addition of 100-fold excess unlabeled wild type Runx (R)
consensus binding site, and its migration was hindered by
Runx2-specific antisera. The Runx2 complex was not altered by anti-HA
antibodies or by unlabeled oligonucleotides containing a scrambled
Runx-binding site or the wild type LEF1-binding site. These data
demonstrate that LEF1 specifically interacts with the osteocalcin
promoter.

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Fig. 1.
LEF1 binds the mOG2 promoter.
A, multiple sequence alignment indicates that the sequence
and positioning of Runx- and LEF1-binding sites are conserved in the
osteocalcin promoters of mouse, rat, and human (mOG1, NCBI
accession number 4204979; mOG2, NCBI accession number 4204980; rat,
NCBI accession number 205865; human, NCBI accession number 22947853).
The Runx and LEF1 recognition sites are boxed.
Dashes represent conserved nucleotides. The sequence of the
mOG2 EMSA oligonucleotide probe ( 144 to 110) is depicted in the
multiple sequence alignment. B, LEF1 interacts with the mOG2
promoter. Electrophoretic mobility shift assay using the wild type EMSA
oligonucleotide and 5 µg of protein from COS cell lysates expressing
either LEF1-HA (left panel) or Runx2 (right
panel). LEF1 complexes are indicated with arrows. Runx2
complexes are marked with arrowheads. Cold competitors
containing wild type Runx consensus site (R), wild type LEF1
consensus site (L), mutant Runx-binding site
(mtR), and mutant LEF1 binding site (mtL) were
added in 100-fold excess. Anti-Runx2 or anti-HA (LEF1) antibodies were
added at 240 µg/ml. Free probe is visible at the bottom of
each panel.
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LEF1 Inhibits Runx2-dependent Activation of the mOG2
Promoter--
Because the LEF1- and Runx2-binding sites are separated
by only four base pairs, we examined the effects of LEF1 on the
transcriptional activity of Runx2. MC3T3 (murine pre-osteoblasts),
C3H10T1/2 (murine mesenchymal precursors), or UMR-106 (rat
osteosarcoma) cells were transiently transfected with the
mOG2-luciferase reporter in the presence or absence of Runx2 and/or
LEF1 expression plasmids. Consistent with data previously reported by
others (37, 40), Runx2 proteins with amino-terminal sequences of either
MRIPV or MASNSL activated the mOG2 promoter ~4-fold in MC3T3 cells
(Fig. 2). LEF1 did not significantly
alter the basal activity of the wild type mOG2 reporter. However, LEF1
blocked the activation induced by either Runx2 isoform (Fig.
2A). LEF1 similarly repressed Runx2-dependent
activation of mOG2 in osteoblast precursor C3H10T1/2 cells (Fig.
2B) and in UMR-106 osteosarcoma cells (Fig. 2C).
The inhibition was dependent on the amount of LEF1 expression plasmids added to UMR-106 and MC3T3 cells (Fig. 2, C and
D). Complete inhibition was observed when equal amounts of
LEF1 and Runx2 expression plasmids were co-transfected into these
cells. Importantly, co-expression of LEF1 did not suppress Runx2
protein levels (Fig. 2E). These data indicate that LEF1
represses Runx2 activity in osteoblasts, thereby defining a novel
intermolecular mechanism that may regulate Runx2 activity during
differentiation and a functional role for LEF1 in osteoblasts.

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Fig. 2.
LEF1 represses Runx2-mediated mOG2-luc
transcription. A, LEF1 represses activation of the
osteocalcin promoter by type I (MASNS) and type II (MRIPV) Runx2
isoforms in preosteoblasts. MC3T3 cells were transfected with mOG2-luc
(300 ng), pCMV-SEAP (500 ng), and pCMV5-Runx2 (MRIPV) (500 ng) and/or
pCMV5-LEF1-HA (500 ng) as indicated. Normalized luciferase is
represented as fold activation over control. The luciferase values
represent the means of triplicate samples ± S.E. B,
LEF1 represses Runx2-mediated activation in mesenchymal precursors.
C3H10T1/2 cells were transfected with mOG2-luc (300 ng), pRL-TK (50 ng), and pCMV5-Runx2 (MASNS) (500 ng) and/or pCMV5-LEF1-HA (500 ng) as
indicated. C, LEF1-mediated repression of Runx2 is
concentration-dependent. MC3T3 cells were transfected as in
A but with increasing concentrations of pCMV5-LEF1-HA as
indicated. D, UMR-106 cells were transfected with mOG2-luc
reporter plasmid (100 ng), pRL-TK (5 ng), pCMV5-Runx2 (MRIPV) (100 ng),
and increasing concentrations of pCMV5-LEF1-HA as indicated.
E, LEF1 does not decrease Runx2 expression. COS cells were
transiently transfected with pCMV5-LEF1-HA and/or pCMV5-Runx2 (MRIPV).
Whole cell extracts were resolved by SDS-PAGE. Runx2 and LEF1 were
detected by immunoblotting with Runx2 and HA antisera,
respectively.
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The LEF1-binding Site in mOG2 Is a Repressive Element but Is Not
Required for Repression of Runx2 Activity--
To determine the
requirement of the LEF1-binding site in osteocalcin regulation, we made
point mutations in the mOG2 EMSA probes and luciferase reporter
constructs that disrupt the binding sites for LEF1, Runx2, or both
factors (Fig. 3A). The
mutations completely eliminated DNA binding of the respective
transcription factor(s) without affecting the DNA binding ability of
the other (Fig. 3B). Mutations in the Runx2-binding site
decreased basal activity of mOG2 (Fig. 3C). This result is
consistent with reports from other laboratories (37, 40).
Interestingly, mutations in the LEF1-binding site increased the basal
activity of the mOG2-luciferase reporter in MC3T3 cells by 2-3-fold
(Fig. 3C). Immunoblot and in situ
immunofluorescence analyses indicated that MC3T3 cells express LEF1 in
their nuclei (Fig. 3, D and E). These data
indicate that endogenous LEF1 suppresses the basal activity of mOG2 in osteoblasts. This is consistent with the role of LEF1 as a
transcriptional repressor in the absence of Wnt signals (22).

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Fig. 3.
The LEF1 site is not required for
LEF1-mediated transcriptional repression. A, schematic
of mutations introduced into the mOG2 reporter plasmids and EMSA probes
by site-directed mutagenesis. The indicated changes were made in either
the Runx recognition sequence or LEF1 recognition sequence by
site-directed mutagenesis. Dashes represent nonmutated
nucleotides. Electrophoretic mobility shift assay probes were
synthesized to correspond to the mutations made in the reporter
plasmids. B, mutations in the Runx2- and LEF1-binding sites
specifically eliminate binding of the respective transcription factor.
The indicated wild type or mutant radiolabeled probes were incubated
with COS lysates expressing Runx2 or LEF1-HA. Runx2 complexes are
marked with arrowheads; LEF1-HA complexes are marked with
arrows. C, deletion of the LEF1-binding site
increases the basal activity of the mOG2 promoter. MC3T3 cells were
transfected as described for Fig. 2A using the indicated
mOG2-luc mutants. The normalized luciferase values are represented as
fold activity over wild type mOG2-luc. D, LEF1 is detectable
as a doublet in MC3T3 cells. MC3T3 cell lysates were resolved by
SDS-PAGE and immunoblotted with anti-LEF1 or anti- -catenin
antibodies. E, LEF1 resides in the nucleus of MC3T3 cells.
MC3T3 were grown on coverslips and fixed in paraformaldehyde. The cells
were lysed with 0.3% Triton X-100 in PBS. LEF1 and -catenin were
detected with their respective monoclonal primary antibody followed by
a goat anti-mouse secondary antibody conjugated to Alexa-546. The
proteins were visualized with fluorescence microscopy. WT,
wild type.
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We next utilized the mOG2 mutant reporter constructs to begin to
determine the mechanism by which LEF1 represses Runx2. MC3T3 cells were
transiently transfected with wild type or mutant mOG2 reporters in the
presence or absence of Runx2 and LEF1 expression plasmids. Because
basal activities of the mutant reporter constructs differed (Fig. 3),
the data were normalized to the activity of the indicated mOG2 reporter
in the absence of Runx2 and LEF1 expression plasmids (Fig.
4). As previously demonstrated, Runx2
activates the wild type mOG2 promoter ~3-fold (Fig. 4A,
top panel). The Runx2-binding site is necessary for Runx2
activation (Fig. 4A, second and fourth panels
from top). However, mutations in the LEF1-binding site did
not significantly affect Runx2-dependent activation (Fig.
4A, third panel from top). Surprisingly, the LEF1-binding site was also not required for LEF1-mediated repression of
Runx2 (Fig. 4A, third panel from top). To confirm
that LEF1-binding sites are not necessary for repression, we tested the
effects of LEF1 on Runx2-driven expression of an artificial promoter, p6OSE2 (37), which contains six Runx-binding elements upstream of the
minimal osteocalcin promoter (
34/+13) and luciferase, and has no
recognizable LEF1-binding sites. Runx2 activated the p6OSE2 reporter by
more than 10-fold (Fig. 4B). LEF1 did not affect the basal
activity of the reporter but repressed Runx2-induced activation
~4-fold. In contrast, LEF1 did not inhibit GAL or GAL-VP16 activity
on a GAL-responsive promoter, pAH205 (Fig. 4C). Thus, LEF1
specifically inhibits Runx2, and the LEF1 DNA binding is not required
for repression of Runx2-dependent activation of the mOG2
promoter.

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Fig. 4.
LEF1-binding site is not required for
LEF1-mediated repression of Runx2. A, LEF1 represses
Runx2-mediated activation of a mOG2-luc reporter lacking the LEF1 DNA
binding site. MC3T3 cells were transfected with 300 ng each of the
indicated mOG2-luc reporter, pCMV-SEAP (500 ng), and pCMV5-Runx2
(MRIPV) (500 ng) and/or pCMV5-LEF1-HA (500 ng) as indicated. Specific
mutations are shown in Fig. 3A. In the schematic on the
left, black boxes represent Runx2-binding sites
(R), and open boxes represent LEF1-binding sites
(L). B, LEF1 represses Runx2-mediated activation
of a Runx-responsive synthetic reporter. MC3T3 cells were transfected
in 6-well plates with 300 ng of p6OSE2-luc, 500 ng of pCMV-SEAP, and
500 ng of pCMV5-Runx2 and/or pCMV5-LEF1-HA. Luciferase values were
normalized to SEAP activity and represented as fold activation over
p6OSE2-luc alone. A schematic of the p6OSE-luc reporter is represented
above. Runx-binding sites (R) are indicated with white
boxes. C, LEF1 repression is specific to Runx2-mediated
activation. MC3T3 cells were transfected with pAH205-luc (300 ng),
Gal-VP16 (300 ng), and/or pCMV5-LEF1-HA (500 ng). Gal DNA-binding sites
(G) are represented as white boxes.
WT, wild type.
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LEF1 Interacts with the RHD--
To determine how LEF1 is
suppressing Runx2, we considered five potential mechanisms of
transcriptional repression: 1) competition for DNA binding sites, 2)
squelching of the basal transcriptional machinery, 3) quenching of the
activation properties of DNA-bound Runx2, 4) indirect transcriptional
repression by complexing with Runx2 and preventing DNA binding, and 5)
co-repressor recruitment (41-43). Competition is unlikely because a
DNA probe containing just the Runx-binding element does not compete
with a mOG2 probe for LEF1 (Fig. 1B), and LEF1 does not bind
to a Runx2-specific probe (data not shown). Squelching also does not
appear to be the mechanism because ectopic LEF1 did not repress the
basal activity of the wild type mOG2 promoter (Fig. 2). To further
explore the possibility that LEF1 quenches or indirectly represses
Runx2, we asked whether LEF1 physically interacts with Runx2. Using a panel of Runx2 and Runx1 GST fusion proteins (Fig.
5A), we found that LEF1
interacts with the amino termini of both Runt domain proteins. The Runt
homology domain (RHD; Runx1 residues 50-179), which
mediates DNA binding, is necessary and sufficient to interact with
full-length LEF1 in vitro (Fig. 5B). A single
point mutation, L148D, in the Runx1 RHD that eliminates DNA binding
(44) also prevented LEF1 interactions. We then mapped the Runx-binding
site in LEF1 with a panel of LEF1 truncation mutants (Fig.
5A). Neither the
-catenin-binding domain nor the extreme
carboxyl terminus was necessary for LEF1 to interact with the RHD
(residues 50-179) (Fig. 5C). The HMG domain, however, was
required and sufficient to interact with the RHD. This interaction
persisted in the presence of ethidium bromide, indicating that
intermolecular association between respective DNA-binding domains of
LEF1 and Runx2 is not mediated by DNA present in the reactions (Fig.
5C, right panel). These data demonstrate a novel
interaction between the nuclear LEF1 and Runx2 and suggest that LEF1
indirectly represses Runx2 activity by binding its DNA-binding
domain.

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Fig. 5.
LEF1 binds the RHD of Runx2 and Runx1.
A, schematic representation of the LEF1, GST-Runx2, and
GST-Runx2 proteins used in protein interaction assays. -cat
BD, -catenin-binding domain; CAD,
trans-activation domain; TLE BD, transducin-like
enhancer of split binding domain; NLS, nuclear localization
signal; AD, activation domain. B, LEF1
specifically binds the RHD of Runx2 and Runx1. In vitro
transcribed and translated, 35S-radiolabeled full-length
LEF1 was incubated with the indicated GST-Runx fusion proteins.
Proteins interacting with GST or GST-Runx2 or 20% of the
35S-LEF1 input were resolved by SDS-PAGE and detected by
autoradiography. C, the HMG domain is necessary and
sufficient to interact with the RHD. GST pull-downs mapping the Runx
interaction site on LEF1. Ethidium bromide (EtBr, 50 µg/ml) was added to some reactions to disrupt DNA-mediated binding.
The arrowhead indicates the appropriately sized band
representing LEF1 HMG.
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The HMG and
-Catenin-interacting Domains of LEF1 Contribute to
Repression of Runx2--
To determine the regions of LEF1 required for
repressing Runx2 activity and to test the fifth potential mechanism of
repression (i.e. co-repressor recruitment), we assayed the
LEF1 and Runx2 truncation mutants in the mOG2 transcription assay. The
LEF1 and Runx2 proteins used in these experiments are diagrammed in
Fig. 6A. All LEF1 and Runx2
proteins localized to cell nuclei (data not shown). The LEF1-HMG
domain, which interacts with Runx2, was necessary for repression of
Runx2 transcriptional activity (Fig. 6B). The HMG and a LEF1
protein lacking the extreme carboxyl terminus (
C) also partially
blocked Runx2-dependent activation but was not as effective
as full-length LEF1 (Fig. 6C). These results suggest that
additional regions of LEF1 are required for maximal repression. This
hypothesis was supported by experiments with a LEF1 protein lacking the
-catenin binding domain (
-cat), which did not repress
Runx2-dependent transcription (Fig. 6B). Thus,
the HMG is required for LEF1 to repress transcription, but the
-catenin-binding domain also contributes to repression.

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Fig. 6.
LEF1 HMG domain and
-catenin-binding domain are required for full
repression. A, schematic of the LEF1 and Runx2 proteins
used for transcriptional assays. B, the LEF1-HMG and
-catenin-binding domains are necessary for complete repression of
Runx2. MC3T3 cells were transfected with mOG2-luc (300 ng), pCMV-SEAP
(500 ng), pCMV5-Runx2 (MASNS) (500 ng), and/or 500 ng of the indicated
pCMV-Gal-LEF1 protein. The luciferase values were normalized to SEAP
activity. The effects of the indicated LEF1 protein on Runx2 activity
are plotted. C, the HMG domain is not sufficient for full
repression by LEF1. MC3T3 cells were transfected as in B
with the indicated LEF1 plasmids. Luciferase values were normalized to
SEAP activity and expressed as fold activation of mOG2-luc alone.
D, the TLE-binding domains of Runx2 and LEF1 are not
required for LEF1-mediated repression of the osteocalcin promoter.
MC3T3 cells were transfected as in B. The luciferase values
were normalized to SEAP activity and are depicted as fold activation
over mOG2-luc.
|
|
Both Runx2 and LEF1 bind the TLE co-repressors (45). To determine
whether LEF1 interacts with TLE proteins that are recruited to the
promoter by the Runx2 carboxyl terminus to repress transcription, we
determined the effects of LEF1 proteins on a truncated Runx2 protein
(1-498) that lacks the TLE-binding domain. Runx2 (1-498) retains the
DNA binding and activation domains and thus activated mOG2 by 5-fold
(Fig. 6D). LEF1 and LEF1-HMG had similar effects on Runx2
(1-498) as they did on wild type Runx2. Thus, LEF1 completely blocked
Runx2 (1-498) activity, and the LEF1-HMG, which lacks the TLE-binding
domain, partially repressed it (Fig. 6D). These data
indicate that TLE recruitment is not required for LEF1-mediated repression of Runx2; however, the possibility that TLE may contribute to LEF1-mediated repression of Runx2 cannot be excluded.
Gain-of-function
-Catenin Enhances LEF1-mediated Repression of
Runx2--
Because LEF1 mutants lacking the
-catenin-binding domain
were less effective at inhibiting Runx2 than wild type LEF1, we hypothesized that
-catenin may participate in LEF1-mediated
repression of Runx2. To test this hypothesis, we co-transfected an
expression plasmid for a gain-of-function (GOF)
-catenin. This
cDNA contains point mutations that are found in cancer cells and
that prevent
-catenin from being degraded, thereby increasing its
levels and facilitating its transport into the nucleus (46, 47).
Interestingly, GOF
-catenin augmented the basal activity of the mOG2
promoter in a concentration-dependent manner (Fig.
7). In contrast, GOF
-catenin did not
affect Runx2-dependent activation of the mOG2 promoter, but
it increased LEF1-mediated repression of Runx2 in a
concentration-dependent manner. These data suggest that
LEF1 and
-catenin cooperatively repress Runx2-dependent
activation. In addition, the observation that GOF
-catenin activates
mOG2 suggests that
-catenin activation may stimulate this promoter via multiple mechanisms.

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|
Fig. 7.
Constitutively activated
-catenin increases LEF1- mediated repression of
Runx2. Constitutively activated -catenin enhances LEF1-mediated
repression in a concentration-dependent manner. MC3T3 cells
were transfected with mOG2-luc (300 ng), pCMV-SEAP (500 ng), and
pCMV5-Runx2 (MPIRV) (500 ng) and/or the indicated amounts of
constitutively activated -catenin plasmid (GOF
-catenin).
|
|
 |
DISCUSSION |
Runx2 is a crucial transcription factor for osteoblast
development. Runx2 expression and activity are controlled by
transcriptional, translational, and post-translational mechanisms (9).
Runx2 activity is further modulated by interactions with other
proteins, including co-activators, co-repressors, and other
transcription factors (13-16). Genetic alterations that decrease Runx2
expression or cause structural changes demonstrate that Runx2 is
required for osteoblast development from mesenchymal stem cells, the
terminal differentiation of committed osteoblasts to osteocytes, and
the expression of late osteoblast genes like osteocalcin (2, 3, 6, 48,
49). Runx2 is expressed in proliferating osteoblasts; however, it does
not appear to play a crucial role in regulating osteoblast number (2,
3, 5). This suggests that Runx2 activity may be suppressed or
controlled in proliferating osteoblasts. Transgenic models support this
hypothesis because increased expression of Runx2 in proliferative
osteoblasts causes osteopenia and bone fractures (7, 8). The mechanisms
regulating Runx2 activity during osteoblast growth phases are not understood.
In this report we describe novel functional and physical interactions
between Runx2 and LEF1 that may contribute to the regulation of Runx2
activity in osteoblasts. LEF1 is a nuclear effector of the canonical
Wnt signaling pathway, which is commonly thought to convert LEF1 to an
activator by interactions with
-catenin (22, 23). We identified a
functional LEF1 recognition sequence in close proximity to the crucial
Runx-binding site in the osteocalcin promoter. The LEF1 site is a
repressive element, and ectopic expression of LEF1 proteins inhibited
Runx2-dependent activation of the osteocalcin promoter in
osteoblast lineage cells. This result is consistent with LEF1 being a
repressor of transcription. We initially hypothesized that activation
of the canonical Wnt signaling pathway would reverse LEF1-mediated
repression. Surprisingly, a constitutively active
-catenin mutant
that mimics Wnt activation did not prevent LEF1-mediated repression and
in fact enhanced suppression. These data suggest that activation of
LEF1 by Wnt signaling represses Runx2-dependent activation of the osteocalcin promoter. Recent data from developmental models indicate that the Wnt signaling pathway indeed directly represses transcription of some genes (27-29, 50-52). Interestingly, LEF1-mediated repression of Runx2 on the osteocalcin promoter is
remarkably similar to Wg (Wnt homologue)- and dTCF (LEF1)-mediated repression of Drosophila stripe. Thus, Wg and dTCF repress
Cubitus interruptus-mediated activation of the stripe
promoter where the dTCF-binding site is only four base pairs from a
crucial Cubitus interruptus recognition sequence (29). These data
indicate that repression mechanisms used by LEF1/TCF factors may be conserved.
The mechanisms by which Wnt signaling pathways repress transcription
are not as clearly defined. Our data suggest that LEF1 indirectly
represses transcription by masking the DNA-binding domain of Runx2. We
found that LEF1 interacts with Runt domain proteins and that
LEF1-binding sites in DNA are not necessary for repression. LEF1 did
not repress GAL-VP16-dependent activation of the
GAL-responsive promoter. Thus, LEF1 specifically inhibits the action of
Runx2 (indirect repression) but does not repress transcription by
interacting with the basal transcriptional complex (direct repression).
This is a potentially powerful means of repression because it predicts
that a LEF1-binding site is not necessary to suppress Runx2
trans-activity. Alterations in LEF1 expression levels could
therefore regulate Runx2 activity in osteoblasts. Interestingly, LEF1
mRNA levels are decreased in osteoblasts lacking the Wnt receptor
LRP5 (33). These data suggest that an autofeedback loop may regulate
LEF1 expression in osteoblasts. Additional data in support of this
autofeedback mechanism are that the canonical Wnt signaling pathway
stimulates LEF1 expression (53). High levels of the Wg (Wnt) receptor
Fzd2 stabilize Wg in Drosophila which, in turn decrease Fzd2
expression (54, 55). Thus, local concentrations of Wnts and/or soluble
Wnt antagonists may autoregulate intracellular signals and gene
transcription in vivo.
The physical and functional interactions between LEF1 and Runx2 may
link two pathways that are crucial for osteoblast maturation. As
described above, Runx2 is required for terminal osteoblast differentiation. The role of LEF1 in osteoblasts has not yet been directly studied; however, LEF1 is expressed in these cells and is thus
a potential nuclear effector of the Wnt signaling pathway in
osteoblasts. Mutations in the Wnt receptor LRP5 have significant effects on bone density. Autosomal recessive and inactivating mutations
in LRP5 decrease bone density in mice and humans (32, 33, 56). In
contrast, activating mutations in LRP5 cause autosomal dominant high
bone mass traits (34, 35, 59). The effects of these mutations on Runx2
activity have not yet been determined. Because LRP5 activates the
canonical Wnt pathway and stabilizes
-catenin (60), which enhances
LEF1-mediated repression of Runx2, it is possible that Wnt signals
transmitted through LRP5 may regulate Runx2 activity.
Our data identify LEF1 as a post-transcriptional regulator of Runx2.
This is an underexplored mechanism of controlling the activity of a
transcription factor crucial for bone development. Previous studies
have identified numerous proteins that either enhance Runx2 function at
the post-transcriptional level (13-16) or modulate Runx2 activity by
regulating the expression/transcription of the Runx2 promoter (61-65).
Our data indicate that additional mechanisms that negatively regulate
Runx2 at the post-transcriptional level also exist.
Repression of Runx2-dependent trans-activation
of the osteocalcin is likely only one consequence of LEF1 expression
and/or
-catenin activation in osteoblasts. In the absence of ectopic LEF1 or Runx2, GOF
-catenin activated the osteocalcin promoter. These data indicate that
-catenin may regulate osteocalcin
expression via multiple mechanisms. It remains to be determined whether
the LEF1-independent
-catenin activation mimics LRP5 activation or whether other stimuli regulate
-catenin function. In addition to the
osteocalcin promoter, we have identified potential LEF1 recognition
sites in other Runx2-regulated osteoblast promoters (e.g.
bone sialoprotein and collagenase 3) (data not shown). Studies that
focus on how Wnt, LRP5,
-catenin, and LEF1 regulate
Runx2-dependent and -independent transcription
of these genes are necessary to gain a full understanding of the
effects of these proteins on gene expression and the maturation of
osteoblasts. It is also important to consider the possibility that LEF1
may respond to Wnt-independent stimuli (e.g. Notch) (66) in
osteoblasts. LEF1 was previously shown to facilitate
Runx1-dependent activation of the T cell receptor enhancer
in a
-catenin-independent manner (67, 68). Finally, the activities
of Runx factors and LEF1 are regulated by interactions with the nuclear
matrix (57, 58). Thus, in addition to extracellular stimuli, gene
structure and tissue-specific factors are also likely to influence how
and when LEF1 and Runx factors interact in vivo.
We thank Drs. Scott Hiebert, Gary Stein,
Rudolf Grosschedl, Randall Moon, Jeffrey Miller, Jennifer Hall, Vivian
Bardwell, and Gerard Karsenty for kindly providing expression
plasmids. We thank Xiaodong Li for technical assistance.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M211443200
The abbreviations used are:
LEF1, lymphoid
enhancer-binding factor 1;
HMG, high mobility group;
GAL, GAL4 DNA
binding domain;
GST, glutathione S-transferase;
HA, hemagglutinin;
GOF, gain-of-function;
EMSA, electrophoretic mobility
shift assay;
RHD, runt homology domain;
LRP5, low density lipoprotein
receptor-related protein 5;
mOG2, mouse osteocalcin 2;
PBS, phosphate-buffered saline;
SEAP, secreted alkaline phosphatase;
MES, 4-morpholineethanesulfonic acid;
TLE, transducin-like enhancer of
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