Osteogenic Protein-1 Up-Regulation of the Collagen X Promoter Activity Is Mediated by a MEF-2-Like Sequence and Requires an Adjacent AP-1 Sequence
Shun-ichi Harada,
T. Kuber Sampath,
Jane E. Aubin and
Gideon A. Rodan
Department of Bone Biology and Osteoporosis Research (S.H.,
G.A.R.) Merck Research Laboratories, West Point, Pennsylvania
19486
Creative Biomolecules, Inc. (T.K.S.) Hopkinton,
Massachusetts 01748
Department of Anatomy and Cell Biology
(J.E.A.) Faculty of Medicine University of Toronto Toronto,
Ontario M5S 1A8, Canada
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ABSTRACT
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Bone morphogenetic proteins induce chondrogenesis
and osteogenesis in vivo. To investigate molecular
mechanisms involved in chondrocyte induction, we examined the effect of
osteogenic protein (OP)-1/bone morphogenetic protein-7 on the collagen
X promoter. In rat calvaria-derived chondrogenic C5.18 cells, OP-1
up-regulates collagen X mRNA levels and its promoter activity in a cell
type- specific manner. Deletion analysis localizes the OP-1 response
region to 33 bp (-310/-278), which confers OP-1 responsiveness to
both the minimal homologous and heterologous Rous sarcoma virus
promoter. Transforming growth factor-ß2 or activin, which
up-regulates the expression of a transforming growth
factor-ß-inducible p3TP-Lux construct, has little effect on collagen
X mRNA and on this 33-bp region. Mutational analysis shows that both an
AP-1 like sequence (-294/-285, TGAATCATCA) and an A/T-rich myocyte
enhancer factor (MEF)-2 like sequence (-310/-298, TTAAAAATAAAAA) in
the 33-bp region are necessary for the OP-1 effect. Gel shift assays
show interaction of distinct nuclear proteins from C5.18 cells with the
AP-1-like and the MEF-2- like sequences. OP-1 rapidly induces nuclear
protein interaction with the MEF-2-like sequence but not with the AP-1
like sequence. MEF-2-like binding activity induced by OP-1 is distinct
from the MEF-2 family proteins present in C2C12 myoblasts, in which
OP-1 does not induce collagen X mRNA or up-regulate its promoter
activity. In conclusion, we identified a specific response region for
OP-1 in the mouse collagen X promoter. Mutational and gel shift
analyses suggest that OP-1 induces nuclear protein interaction with an
A/T-rich MEF-2 like sequence, distinct from the MEF-2 present in
myoblasts, and up-regulates collagen X promoter activity, which also
requires an AP-1 like sequence.
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INTRODUCTION
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Bone morphogenetic proteins (BMPs) are members of the
transforming growth factor (TGF)ß superfamily and play fundamental
roles in the development of Drosophila, Xenopus,
and mammals (1). Osteogenic protein (OP)-1/BMP-7 belongs to the 60A
subgroup of the BMP family, which has a 75% homology in the mature
carboxyl-terminal domain and includes BMP-5, 6, and 8 (1, 2). By
in situ hybridization, OP-1 mRNA is expressed in kidney,
heart, eye, and growth plate, as well as other sites (3). Mice lacking
OP-1/BMP-7 display severe defects in kidney and eye development and
have skeletal abnormalities in the rib cage, skull, and hindlimbs (4, 5). Despite a high level of expression, no consistent defects have been
observed in the axial skeleton, possibly due to compensation by other
BMPs (3, 6).
During endochondral bone formation, chondrocytes proliferate,
differentiate, hypertrophy, and express specific matrix proteins
including collagen type II, IX, XI, and X, which is selectively
expressed in hypertrophic cartilage (7, 8). Collagen X is a homotrimer
of three 59-kDa
1(X) chains. The collagen X gene has only three
exons, the last one encoding the entire triple helical collagenous
domain. The 5'-regulatory region of the
1 collagen X gene has been
characterized in chicken, mouse, calf, and human (9, 10, 11, 12).
Transcriptional regulation of the collagen X promoter has been analyzed
primarily for the chicken gene. In transgenic mice, 4700 bp of the
chicken collagen X promoter induced tissue-specific expression of a
dominant negative collagen X mutant in hypertrophic chondrocytes,
resulting in spondylometaphyseal dysplasia (13). In vitro
analysis of the cis-acting elements in the chicken collagen
X gene identified cell type-specific multiple repressor regions that
are active in fibroblasts and immature chondrocytes, but not in
hypertrophic chondrocytes (14). In addition, a recent study, using
in vivo footprinting and in vitro reporter
assays, suggests that the proximal promoter region plays a role in the
specific expression of chicken collagen X in hypertrophic chondrocytes
(15).
BMPs induce the expression of phenotypic markers for chondrocytes and
osteoblasts in bone marrow cells, limb bud cells, rat calvaria cells,
and myoblastic cells in vitro (16, 17, 18, 19). However, the
molecular mechanisms for these effects are not well characterized.
RCJ3.1C5.18 (C5.18) is a spontaneously immortalized chondrogenic
subclone (20) of the fetal rat calvaria-derived pluripotential RCJ 3.1
cell line, which gives rise to myotubes, adipocytes, chondrocytes, and
osteoblasts in vitro (21). We found that, in C5.18 cells,
OP-1 stimulates proliferation and induces the expression of type II
collagen at 12 h and type X collagen at 24 h, type I collagen
at 48 h, and alkaline phosphatase and osteocalcin at 72 h
(22). OP-1 also induces, in these cells, mRNAs for N-cadherin, neural
cell adhesion molecule (23, 24, 25), and Msx-2 (26), which have been shown
to be induced by BMP in vivo and/or in vitro in
undifferentiated mesenchymal cells (22), suggesting that C5.18 cells
differentiate into chondrocytes and/or osteoblasts in response to
OP-1.
To investigate the molecular basis for OP-1 induction of chondrocyte
phenotypic markers, we examined the effect of OP-1 on the mouse
collagen X promoter in C5.18 cells. We found that OP-1 induces collagen
X gene expression via a cell type-specific 33-bp response region that
contains an A/T-rich sequence, analogous to a consensus myocyte
enhancer factor (MEF)-2 site, and an AP-1 like sequence. Mutational and
gel shift analyses showed that OP-1 induces nuclear protein interaction
with the MEF-2 like sequence, which is distinct from MEF-2, and
up-regulates promoter activity via a mechanism that also requires an
AP-1-like sequence.
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RESULTS
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OP-1 Stimulates Collagen X Promoter Activity in C5.18 Cells via the
-310/-278 Collagen X Promoter Region
C5.18 is a spontaneously immortalized chondrogenic cell line
derived by several rounds of subcloning from a pluripotent fetal rat
calvaria cell line (20, 21). In these cells, OP-1 (30250 ng/ml)
induces expression of mRNAs for collagen II (22) and collagen X. C5.18
cells also express high levels of mRNAs for ALK3 and ALK6, specific
type I receptors for BMPs (data not shown).
Using a series of 5'-deletion fragments of the collagen X promoter
placed upstream of a promoterless luciferase reporter gene transfected
into C5.18 cells, we found that OP-1 (30250 ng/ml) induces luciferase
activity driven by a 1067-bp (-1008/+59) as well as by a 386-bp
(-327/+59) collagen X promoter, up to 2.5-fold in a dose-dependent
manner (Fig. 1
and Table 1
). A further
42-bp deletion (-285/+59) abolished OP-1 induction, indicating that
the -327/-286 region is required for the OP-1 effect. OP-1 also
stimulated reporter activity driven by the 951-bp (-327/+624) promoter
region containing the first intron (+66/+624) of the mouse collagen X
gene. This induction was abolished by the 5'- deletion, which leaves
the region (-171/+624) (Fig. 1
). OP-1 also up-regulated luciferase
reporter activity driven by the -1008/-96 collagen X promoter region,
which contains only the upstream start site previously reported (10),
but is 20-fold less active than the -1008/+59 construct containing
both start sites (data not shown). The stimulatory effects of OP-1 on
the collagen X promoter were cell type specific and were not seen in
ROS17/2.8 osteoblastic cells, in NIH3T3 fibroblasts (data not shown),
or in C2C12 myoblasts (Fig. 8
).

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Figure 1. Deletion Analysis of OP-1 Up-Regulation of Collagen
X Promoter Activity in C5.18 Cells
Deletion constructs of the mouse collagen X promoter-luciferase
reporter gene were generated as described in Materials and
Methods; a schematic representation of each reporter construct
is shown in the left panel. Twenty four hours after
transfection of C5.18 cells with constructs, the cells were treated
with OP-1(100 ng/ml) in the presence of 15% FBS and were cultured for
an additional 24 h. Luciferase activity was measured as described
in Materials and Methods, and transfection efficiencies
was monitored with SV40LUC in parallel wells. Data are presented as the
relative mean luciferase activity (% SV40LUC) ±SD.
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Figure 8. OP-1 Has No Effect on Collagen X mRNA Levels (A) or
Collagen X Promoter Activity (B) in C2C12 Myoblasts
Left panel, C2C12 cells were treated with OP-1 (100
ng/ml) for 24 h in the presence of 10% FBS. Total RNA (20 µg
per lane) was subjected to Northern blot analysis with a mouse collagen
X cDNA probe. Migration of 28S and 18S ribosomal RNAs are indicated by
arrowheads. For comparison of RNA loading, a human GAPDH
probe was hybridized to the same filter. The figure represents the
results from one of three similar experiments. Right
panel, C2C12 cells were transfected with deletion constructs of
collagen X-luciferase reporter gene as described in Materials
and Methods. Twenty four hours after transfection, the cells
were treated with OP-1(100 ng/ml) in the presence of 10% FBS and were
cultured for an additional 24 h. The data are presented as the
relative mean luciferase activity (% SV40LUC) ±SD.
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To further localize the OP-1-responsive region, a -327/-248
collagen X promoter fragment was placed upstream of the -90/+59
collagen X minimal promoter, which does not respond to OP-1, or of the
heterologous minimal Rous sarcoma virus (RSV) promoter (-51/+35). The
-327/-248 promoter fragment conferred OP-1 responsiveness to both
(Fig. 2A
and 2B
). A further 26-bp
deletion (-301/-248) abolished the OP-1 response (Fig. 2A
). Moreover,
a shorter 33-bp oligonucleotide corresponding to the -310/-278
region, protected by C5.18 cell nuclear extracts in deoxyribonuclease
(DNase) footprinting assays (data not shown), also conferred OP-1
responsiveness to the RSV minimal promoter in a copy number-dependent
and orientation-independent manner, to a similar extent as the
-327/-248 fragment (Fig. 2B
). These findings demonstrate that the
-310/-278 collagen X promoter region acts as an enhancer and is
sufficient for OP-1 up-regulation of the collagen X promoter activity
in C5.18 cells.

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Figure 2. The -327/-248 Collagen X Promoter Region Confers
the OP-1 Responsiveness to the Minimal Collagen X Promoter (A) and to
the Heterologous RSV Promoter (B)
Collagen X promoter fragments were synthesized and inserted upstream of
the minimal collagen X promoter (90COLXLUC) or the heterologous minimal
RSV promoter luciferase construct (RSVLUC) as described in
Materials and Methods. The data are presented as the
relative mean luciferase activity (% SV40LUC in Fig. 2A and %RSVLUC
in Fig. 2B ) ±SD.
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OP-1 but Not TGFß2 or Activin Stimulates Collagen X Promoter
Activity
OP-1 as well as activin belong to the TGFß superfamily whose
members interact with cell surface serine/threonine kinase receptors.
In C5.18 cells, TGFß2 (2 ng/ml) or activin (100 ng/ml) induced the
expression of the TGFß- inducible p3TP-Lux construct (27), by 4-fold
or 2-fold, respectively, but had little effect on the -327/-248
collagen X promoter region fused to the -90/+59 minimal collagen X
promoter (Fig. 3A
). Nor did TGFß2 or
activin increase collagen X mRNA levels in C5.18 cells (Fig. 3B
). In
addition, OP-1 (100 ng/ml) was less effective (50%) than TGFß2 or
activin in stimulating the p3TP-Lux construct. These results show
specific OP-1 effects on collagen X mRNA and promoter activity and
suggest that OP-1 and TGFß2/activin may stimulate gene expression via
distinct mechanisms in C5.18 cells.

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Figure 3. OP-1, but not TGFß2 or Activin, Up-Regulates the
-327/-248 Collagen X Promoter Activity (A) and Collagen X mRNA Levels
(B) in C5.18 Cells
A, C5.18 cells were transfected with SV40LUC, TGFß-inducible
p3TP-Lux, 90COXLUC, and (-327/-248)x290COLXLUC constructs. Twenty
four hours after transfection, C5.18 cells were treated with OP-1 (100
ng/ml), TGFß2 (2 ng/ml), or activin (100 ng/ml) and cultured for an
additional 24 h. The data are presented as the relative mean
luciferase activity (% SV40LUC) ± SD. B, C5.18 cells were
treated with OP-1 (100 ng/ml), TGFß2 (2 ng/ml), or activin (100
ng/ml) for 24 h. Total RNA (20 µg per lane) was subjected to
Northern blot analysis with a mouse collagen X cDNA probe. The
locations of the 28S and 18S ribosomal RNAs are indicated by
arrowheads. For comparison of RNA loading, a human GAPDH
probe was hybridized to the same filter. The figure represents the
results from one of three similar experiments.
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OP-1 Stimulates Collagen X Promoter Activity via an A/T-Rich
Sequence and an AP-1-Like Sequence Present in the -310/-278
Region
The -310/-278 OP-1 response region contains an
AP-1-like sequence (-294/-285, TGAATCATCA) and an A/T-rich sequence
(-310/-300, TTAAAAATAAA) homologous to the response region for MEF-2,
a MADS (MCM1-agamous-ARG-deficiens-SRF) box family transcription
factor. Mutation of the AP-1-like sequence (-294/-285, TGAATCATCA to
TTCCGCATCA; M1) strongly suppressed the enhancer activity
of this region and completely abolished responsiveness to OP-1 (100
ng/ml) and to basic fibroblast growth factor (bFGF; 10 ng/ml), shown to
activate AP-1 and to stimulate the expression of the
(-310/-278)x3-RSVLUC construct in the presence of 1% serum. On the
other hand, mutation of the A/T-rich sequence (-310/-300, TTAAAAATAAA
to TTAACGCGCGA; M2) abolished response to OP-1, but had
little effect on enhancer activity or on the stimulatory effect of bFGF
on the -310/-278 region. Mutation of the intervening sequence
(-299/-295, AAGGG to CCTTG, M3), located between the
AP-1-like and the A/T-rich sequences, suppressed enhancer activity by
up to 50% but had no effect on either OP-1 or bFGF stimulation.
These results show that both the AP-1-like sequence and the
A/T-rich sequence are required for OP-1 up-regulation of collagen X
promoter activity and suggest that OP-1 acts on the A/T-rich sequence,
but AP-1-like sequence is also required. Basic FGF, on the other hand,
acts directly on the AP-1-like sequence. Consistent with this
conclusion, bFGF, but not OP-1, up-regulated the luciferase activity
driven by the consensus AP-1 sequence fused to the
-fibrinogen
minimal promoter (AP1x3-FIBLUC) (Fig. 4
).
Neither OP-1 nor bFGF had an effect on the minimal
-fibrinogen
promoter (data not shown). Furthermore, a mouse c-fos
expression plasmid cotransfected with (-327/-248)x290COLXLUC
increased its expression by 2-fold, and OP-1 caused a further 2-fold
stimulation (Fig. 5
). These results also
suggest that OP-1 acts on the A/T-rich sequence in the -310/-278
region, and not directly on the AP-1 like sequence, to stimulate the
collagen X promoter activity.

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Figure 4. Mutational Analysis of the -310/-278 Collagen X
Promoter Region
Three copies of the wild type or mutant -310/-278 fragments
(lower panel) were inserted upstream of RSVLUC as
described in Materials and Methods. Twenty four hours
after transfection, C5.18 cells were treated with OP-1 (100 ng/ml) or
bFGF (10 ng/ml) in the presence of 1% FBS for an additional 24 h.
The data are presented as the relative mean luciferase activity (%
RSVLUC) ±SD.
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Figure 5. Effect of c-Fos Coexpression on OP-1 Up-Regulation
of the -327/-248 Collagen X Promoter Region
Collagen X promoter luciferase constructs, 90COLXLUC or
(-327/-248)x290COLXLUC, were cotransfected with the
c-fos expression plasmid (pcDNACFOS) or control pcDNA3
plasmid into C5.18 cells. Twenty four hours after transfection, the
cells were treated with OP-1 (100 ng/ml). The data are presented as the
relative mean luciferase activity (% RSVLUC) ±SD.
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The A/T-Rich Sequence and the AP-1-Like Sequence in the -310/-278
Collagen X Promoter Region Bind Distinct Nuclear Factors Present in
C5.18 Cells
To characterize the nuclear proteins that play a role in OP-1
up-regulation of the collagen X promoter, we performed gel shift assays
with oligonucleotide probes of the -310/-278 region with mutations in
the AP-1-like and/or A/T-rich sequences (Fig. 6
). The wild type
-310/-278 probe identified a major retarded band C and two minor
bands A and B, which were all competed by excess cold homologous
oligonucleotide (Fig. 6A
, lanes 2 and 3). An oligonucleotide
corresponding to the consensus AP-1 sequence also competed for band C,
but not bands A or B (Fig. 6A
, lane 4). In contrast, bands A and C, but
not band B, were competed by excess cold -310/-278 oligonucleotide
with a mutation at the A/T-rich sequence (M2) (Fig. 6A
, lane 5). These
results suggest that band C corresponds to a nuclear protein that
interacts with the AP-1-like sequence, and band B to a nuclear protein
that interacts with the A/T-rich sequence. Consistent with this notion,
band B but not band A, which are both identified by the -310/-278
probe mutated in the AP-1 like sequence (M1) (Fig. 6A
; lanes 68), was
absent after the additional mutation in the A/T-rich sequence (M4)
(Fig. 6A
, lanes 911). Furthermore, the nuclear protein interacting
with the AP-1-like sequence (band C) was supershifted with an antihuman
c-Fos antibody, raised against the conserved domain of Fos family
members (Fig. 6B
). This supershift was blocked by the antigenic
peptide, suggesting the involvement of Fos family proteins in this
complex.

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Figure 6. C5.18 Nuclear Proteins Interact with the AP-1-Like
Sequence and the A/T-Rich MEF-2-Like Sequence of the -310/-278
Collagen X Promoter Region. A, Nuclear extracts from C5.18 cells were
incubated with the oligonucleotide probe COLX(-310/-278) (lanes
15), COLX(-310/-278) with a mutation in the AP-1 like sequence (M1;
lanes 68) or COLX(-310/-278) with mutations in the AP-1-like
sequence and in the A/T-rich sequence (M4; lanes 911). Competition
experiments were performed with 400-fold molar excess nonlabeled
oligonucleotides corresponding to COLX(-310/-278) (lane 3), AP-1
(lane 4), COLX(-310/-278) with a mutation in the A/T-rich sequence
(M2; lane 5), COLX(-310/-278)M1 (lane 8) or COLX(-310/-278)M4 (lane
11). B, C5.18 nuclear extracts (lanes 15) were incubated with
COLX(-310/-295)M2 probe in the presence or absence of an anti-c-Fos
antibody (2 µg; lanes 3 and 5) and/or its antigenic peptide (2 µg;
lanes 4 and 5). C, C5.18 nuclear extracts were incubated with the
oligonucleotide probe COLX(-314/-295). Competition experiments were
performed with 200-fold molar excess of the following oligonucleotides:
wild type COLX(-314/-295) (lane 3), mutant COLX(-314/-295) (lane
4), MEF-2 (lane 5), NP (lane 6), OCTA26 (lane 7), OCT (lane 8), or
E-Box (lane 9).
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The A/T-rich sequence (TTAAAAATAAA) resembles the consensus
sequence for MEF-2 (CTAAAAATAAC). Gel shift assays with a shorter
-314/-295 probe showed that the fast migrating band B was as
effectively competed by the MEF-2 consensus oligonucleotide as by
homologous oligonucleotide, but not by oligonucleotides corresponding
to NP, OCTA26, OCT, or E-Box sequences (Fig. 6C
). A slower migrating
band D was less specific and was competed both by homologous
oligonucleotide and by the unrelated OCTA26, OCT, or E-Box
oligonucleotides (Fig. 6C
).
It is noteworthy that the specific complex with the MEF-2-like
-314/-295 sequence (band B) was not competed by a mutant -314/-295
oligonucleotide (TTAAACATAAA), analogous to a mutant MEF-2
sequence (CTAAACATAAC) that does not bind to MEF-2 protein
(Fig. 6C
). These findings suggest that C5.18 nuclear proteins interact
with the A/T-rich sequence (Fig. 6C
, band B), in a manner similar to
MEF-2 binding to its consensus sequence. The -310/-278 region thus
interacts with two distinct nuclear protein complexes present in C5.18
cells: 1) via the AP-1-like sequence (Fig. 6
, A and B; band C) and 2)
via the A/T-rich MEF-2-like sequence (Fig. 6
, A and C; band
B).
OP-1 Stimulation Increases Nuclear Protein Interaction with the
A/T-Rich MEF-2-Like Sequence but Not with the AP-1-Like Sequence
We further examined the effect of OP-1 on nuclear protein
interaction. OP-1 had no effect on C5.18 nuclear protein interaction
with the AP-1-like sequence, tested with the -310/-278 probe mutated
in the A/T-rich sequence (M2) (Fig. 7
, lanes 69), consistent with the mutational analysis (Fig. 4
). In
contrast, treatment of C5.18 cells with OP-1 (100 ng/ml) for 2 or
6 h increased up to 3- to 4-fold band B probed with the
-314/-295 sequence (Fig. 7
, lanes 15). This band is competed by
cold homologous oligonucleotide. OP-1 treatment had no effect on band D
(Fig. 7
, lanes 15), suggesting that OP-1 specifically increases the
level or the affinity of nuclear proteins that interact with the
MEF-2-like A/T-rich sequence (Fig. 7
, band B).

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Figure 7. OP-1 Increases Nuclear Protein Interaction with the
A/T-Rich MEF-2- Like Sequence but Not with the AP-1-Like Sequence of
the -310/-278 Collagen X Promoter Region in C5.18 Cells
Nuclear extracts from C5.18 cells treated with or without OP-1 (100
ng/ml, for 2 or 6 h) were incubated with the COLX(-314/-295)
oligonucleotide probe (lanes 15) or with COLX(-310/-278)M2 mutated
in the A/T- rich sequence (lanes 69). Competition experiments
contained 400-fold molar excess of homologous oligonucleotide (lane
5).
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The C5.18 Nuclear Proteins That Bind the Collagen X A/T-Rich
Sequence Interact with the Consensus MEF-2 Site but Are Distinct from
MEF-2
MEF-2 family proteins, MEF-2A, B, C, and D, belong to the MADS box
transcription factor family and play an important role in skeletal and
cardiac myoblast differentiation (28). OP-1 has no effect on either the
collagen X promoter or collagen X mRNA levels in C2C12 myoblasts, which
highly express MEF-2 proteins (Fig. 8
).
The -314/-295 probe formed specific slowly migrating complexes (Fig. 9A
, band E) with C2C12
nuclear extracts that were competed by cold homologous oligonucleotide
and by the consensus MEF-2 oligonucleotide. However, C2C12 nuclear
extracts did not form the faster migrating complex induced by OP-1 in
C5.18 cells (Fig. 9A
, lane 2, and Fig. 7
, band B), suggesting cell type
specificity for nuclear protein interaction with the A/T-rich sequence.
On the other hand, C2C12 nuclear proteins form an AP-1 complex similar
to that formed by C5.18 nuclear extracts (data not shown). C2C12
nuclear extracts also formed similar slow migrating but more intense
bands with the consensus MEF-2 probe, competed by cold homologous MEF-2
oligonucleotide, but not by the -314/-295 oligonucleotide, nor by the
mutant -314/-295 oligonucleotide (Fig. 9B
, bands E). These results
suggest that the slowly migrating bands E are formed by the endogenous
MEF-2 proteins present in C2C12 myoblasts.

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Figure 9. OP-1 Up-Regulates MEF-2-Like Binding Activity in
C5.18 Cells That Is Distinct from the MEF-2 Present in C2C12
Myoblasts. A, C5.18 nuclear extracts (lane 2) or C2C12
nuclear extracts (lane 36) were incubated with the COLX(-314/-295)
probe. Competition experiments were performed with 400-fold molar
excess of the following oligonucleotides: wild type COLX(-314/-295)
(lane 4), MEF-2 (lanes 5), COLX(-314/-295)M with a point mutation
(lanes 6). B, C5.18 (lane 2) or C2C12 (lanes 36) nuclear extracts
were incubated with the consensus MEF-2 probe. Competition experiments
were performed with 400-fold molar excess of the following
oligonucleotides; MEF-2 (lane 4), COLX(-314/-295) (lane 5),
COLX(-314/-295)M (lane 6). C, C5.18 nuclear extracts were incubated
with the MEF-2 probe. Competition experiments were performed with
80-fold or 400-fold molar excess of the following oligonucleotides:
MEF-2 (lanes 3 and 4), wild type COLX(-314/-295) (lanes 5 and 6),
COLX(-314/-295)M (lanes 7 and 8).
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The MEF-2 oligonucleotide probe also formed complexes with C5.18
nuclear extracts (Fig. 9C
, bands B). These are similar to the fast
migrating complexes formed with the collagen X A/T-rich sequence (Fig. 6C
and Fig. 7
, band B) and are distinct from those formed by C2C12
nuclear extracts and presumably do not include muscle MEF-2 proteins.
The C5.18 nuclear protein interaction with the MEF-2 probe (Fig. 9C
, bands B) was competed not only by cold MEF-2 homologous oligonucleotide
but also, to a lesser extent, by the COLX(-314/-295) oligonucleotide,
but not by the mutant COLX(-314/-295) oligonucleotide. These results
suggest that the OP-1-inducible MEF-2-like binding activity present in
C5.18 cells, which interacts with the collagen X A/T-rich sequence and
the MEF-2 consensus sequence, is distinct from MEF-2A, B, C, or D,
present in C2C12 cells.
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DISCUSSION
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In this study, we identified a 33-bp response region for OP-1 in
the mouse collagen X promoter that mediates OP-1 up-regulation of type
X collagen in C5.18 cells.
The 33 bp OP-1 response region contains an AP-1 like sequence and a
MEF-2- like A/T-rich sequence that are both required for the OP-1
effect. Gel shift analysis shows that the C5.18 nuclear protein
interaction with the AP-1-like sequence involves a c-Fos family member.
Fos family proteins have previously been implicated in chondrogenesis
and osteogenesis (29). c-fos and its family member, fra-2,
are highly expressed in bone and cartilage during development (30, 31),
and transgenic mice overexpressing a c-fos transgene develop
osteogenic and chondrogenic tumors (29). Expression of c-fos
is induced in osteoblasts, osteocytes, chondrocytes, or bone tissue by
osteogenic factors, mechanical stress, or during fracture repair
(32, 33, 34). BMP-2 and 3 have been shown to increase c-fos mRNA
levels in MC3T3-E1 osteoblastic cells (35). However, several
observations presented here suggest that the induction or activation of
c-Fos itself is not the primary mechanism for OP-1 stimulation of
collagen X promoter activity. First, a mutation of the 33-bp collagen X
promoter OP-1 response region in the MEF-2-like A/T-rich sequence (M2)
abolished OP-1 activation but not the effect of bFGF, known to induce
AP-1 in chondrocytes in the presence of low serum (34). Second, OP-1
failed to stimulate reporter activity driven by an AP-1 consensus
sequence, which was induced by bFGF. Third, OP-1 had no effect on C5.18
nuclear protein interaction with the AP-1 like sequence of the collagen
X promoter, while inducing nuclear protein interaction with the
A/T-rich MEF-2-like sequence. However, the AP-1-like sequence is
required to produce the OP-1 effect as indicated by mutational
analysis. AP-1-like sequences or Fos family proteins have been
implicated in the transcriptional regulation by TGFß (36, 37, 38, 39) and
were shown to play a role in the regulation of the osteocalcin and the
type I collagen promoters (40, 41). An AP-1-like sequence similar to
that of the collagen X promoter is found in the rat osteocalcin
promoter region required for the response to BMP-2 (42). BMP-4
induction of dorso-ventral axis formation and of erythroid
differentiation in ventral mesoderm in Xenopus embryos was
recently shown to involve the ras/raf/AP-1 signaling pathway (43).
MEF-2 family proteins belong to MADS Box transcription factor family,
which includes SRF (serum response factor), MCM1, Agamous and
Deficiens, and interact as homo- or heterodimers with specific A/T-rich
response elements (28). Currently, four distinct MEF-2 family proteins
have been isolated and implicated in skeletal and cardiac muscle cell
differen-tiation: MEF-2A/RSRFC/SL-2, MEF-2B/xMEF-2/RSRFR2, MEF-2C,
and MEF-2D/SL-1 (28). They share homology in the MADS box and the
adjacent MEF-2 domains. We found here that mutating the MEF-2 like
sequence in the collagen X promoter abolished OP-1 induction of
promoter activity and nuclear protein binding to this region. Moreover,
nuclear protein interaction with this sequence was specifically induced
by OP-1 in C5.18 cells. OP-1-inducible MEF-2-like binding activity
(OMBA) is not present in C2C12 myoblasts, which express MEF-2A, B, C,
and D. Additional evidence suggests that OMBA is distinct from
MEF-2A-D. C2C12 cells respond to BMP-2 with expression of phenotypic
markers for osteoblasts but not for chondrocytes (19). Interestingly,
OP-1 had no effect on either the collagen X promoter or collagen X mRNA
levels in C2C12 cells (Fig. 8
).
There are several transcription factors that interact with MEF-2-like
A/T- rich sequences and form complexes that migrate faster in gel shift
assays than MEF-2 complexes: MEF-2 related BBF-1 (44), an RSRF-related
A-rich binding factor (45), ATF35 (46), a zinc finger protein, HF-1b
(47), and a homeo box protein, Gtx (48). The homeodomain protein Mhox
and the POU domain protein Oct-1 can also interact with the MEF-2 site
of the
-cardiac myosin heavy-chain gene (49). However, interaction
of the OMBA with the collagen X MEF-2- like sequence is not competed by
excess oligonucleotides OCT or OCTA26 as well as NP, which competes for
the binding of Gtx to the MEF-2 site (48). In addition, a point
mutation of the collagen X MEF-2-like sequence, analogous to a mutation
that abolishes its binding to MEF-2, also abolished OMBA. Taken
together, our results show that OMBA binds to a MEF-2-like sequence
analogous to that recognized by the MEF-2 family and is distinct from
homeodomain or POU domain proteins, including Oct-1, Mhox, and Gtx.
BMPs act via type I and type II serine/threonine kinase receptors (50).
Recently it was found that serine/threonine phosphorylation of MEF-2C
by casein kinase-II or p38 enhances its DNA binding and transcriptional
activity (51, 52). Interestingly, treatment of C5.18 nuclear extracts
with potato acid phosphatase resulted in loss of OMBA (data not shown),
suggesting a possible role for OP-1-induced phosphorylation in its mode
of action. Further studies are necessary to evaluate the role of
phosphorylation in the OP-1 effects on OMBA.
MADS box proteins regulate cell type-specific gene expression
through cooperation with other regulatory factors (28). SRF interacts
with ETS-domain proteins, which bind a site adjacent to the serum
response element in the c-fos promoter (53). MEF-2 binds DNA
cooperatively with myogenic basic helix-loop-helix proteins via
specific interaction between the DNA-binding domains of MEF-2 and the
myogenic basic helix-loop-helix factors (54). Our finding that
up-regulation of collagen X promoter activity by OP-1 requires both
OMBA, induced by OP-1 treatment, and AP-1 is consistent with these
observations.
OP-1 belongs to the TGFß superfamily, which includes TGFß1,
2, 3, and activin (1). Although TGFßs and activin have been
implicated in osteogenesis and in chondrogenesis (55, 56, 57), induction of
endochondral osteogenesis at heterotopic sites in vivo is
unique to BMPs. In cultured cells in vitro, TGFß has
bifunctional effects on chondrocyte differentiation (58, 59, 60). In
C5.18 cells, OP-1, but not TGFß2 or activin, induces collagen X
mRNA and the expression of the OP-1-responsive promoter region.
Transcriptional regulation by TGFß family members has been
examined in the promoters of many genes (36, 37, 38, 39, 61, 62, 63); however, the
regulation of gene expression by BMPs in osteoblasts and chondrocytes
is not well characterized. In C3H10T1/2 cells, BMP-2 induces nuclear
protein binding to the proximal E-box (OCE-1) of the rat osteocalcin
promoter (64), and its activity was shown to be up-regulated by BMP-2
in mouse limb bud-derived MLB13MYC cells (42). However, these studies
did not examine the effect of other TGFß superfamily members. To our
knowledge, the OP-1 response region characterized in this study is the
first specific response region for a BMP member (OP-1) of the TGFß
superfamily.
A genetic approach in Drosophila has led to isolation
of several genes downstream of dpp, a Drosophila
counterpart of BMP-2/4, including Mad (Mothers against
dpp) (65). Isolation of vertebrate homologs of Mad, the Smads
family, demonstrate that distinct Smads respond to distinct subsets of
TGFß family members and convey specific information to the nucleus
(66). Smad1/MADR1, a human homolog of MAD, is phosphorylated by type I
BMP receptor and translocates to nuclei in response to BMP-2 but not to
TGFß (67, 68). In addition, Smads possess transcription activator
domains when fused to the DNA- binding domain of GAL4 (69) and bind to
DNA as a complex with transcription factors (70). A recent report also
shows that Smads could interact directly with specific DNA sequences
(71). Further studies may identify a role for Smad proteins in
chondrocyte and osteoblast differentiation and in the regulation of
their phenotypic markers, such as collagen X. However, the regulatory
sequence responsive to OP-1/BMP-7 described in this study is not
homologous to the reported Smad-binding sequences (70, 71).
In summary, we have identified a specific responsive region for OP-1 in
the collagen X promoter. Functional analysis of this region
demonstrated the requirement for an AP-1-like sequence and a MEF-2-like
sequence that interacts with nuclear factors in response to OP-1.
Further characterization of these nuclear factors and their cooperation
with the AP-1-like sequence should increase our molecular understanding
of BMP action.
 |
MATERIALS AND METHODS
|
---|
Reagents
Restriction enzymes, Klenow fragments, T4 kinase, and T4 ligase
were purchased from New England Biolabs (Beverly, MA). All tissue
culture media and reagents were purchased from GIBCO BRL (Grand Island,
NY), except for FBS, which was obtained from JRH Biosciences (Lenexa,
KS). All chemicals not indicated in Materials and Methods
were purchased from Sigma Chemical Co. (St. Louis, MO).
Oligonucleotides were synthesized by GIBCO BRL.
Mouse Collagen X Promoter-Luciferase Constructs
A 1067-bp fragment (-1008/+59) of the mouse collagen X promoter
region was obtained by PCR, using a GeneAmp Kit and a Thermocycler
(Perkin-Elmer/Cetus, Norwalk, CT), from mouse genomic DNA (CLONTECH,
Palo Alto, PA). PCR was performed with 34-mer oligonucleotide primers
containing a KpnI site (5'-primer) and an MluI
site (3'-primer) based on the published sequence of the mouse collagen
X gene (10). This amplified DNA fragment was purified by agarose gel
electrophoresis, digested with appropriate restriction enzymes, and
subcloned into the KpnI/MluI sites of the
promoterless pGL2-basic luciferase vector (Promega, Madison, WI). The
1067-bp fragment was also sequenced with a Sequenase Version 2.0 DNA
Sequence Kit (United States Biochemical, Cleveland, OH) and was found
to be identical to the published sequence (10). A series of 5'-deletion
fragments of the collagen X promoter were also generated by PCR by
using 27-mer 5'-primers with a KpnI site and the 3' 34-mer
primer with an MluI site positioned at +59 and were
subcloned into pGL2, generating 1008COLXLUC, 816COLXLUC, 725COLXLUC,
482COLXLUC, 327COLXLUC, 301COLXLUC, 285COLXLUC, 171COLXLUC, 132COLXLUC,
and 90COLXLUC. Sequences for these oligonucleotides, described in the
references cited are as follows; NP(5'-TCATTAATTGA-3');
OCTA26(5'-GATCAGTACTAATTAGCATTATAAAG-3');
OCT(5'-GGTAATTTG-CATTTCTAA-3');
E-Box(5'-GATCCCCCCAACACCTGCTGCCTGA-3'). Constructs including the first
intron +66/+624 were also synthesized by PCR using a 3'-34-mer
primer with an MluI site posi-tioned at +624, creating
-327/+624COLXLUC and -171/+624COLXLUC. The numbering of the collagen
X promoter was based on published information (10), with the downstream
transcription start site designated as +1. To generate
(-327/-248)x290COLXLUC or (-310/-248)x290COLXLUC, a -327/-248
or a -310/-248 fragment of the collagen X promoter was obtained by
PCR with 5'-primers containing a KpnI site and a 3' 27-mer
primer with a KpnI site positioned at -248 and subcloned
into the KpnI site of 90COLXLUC. The RSV promoter luciferase
vector (RSVLUC) contains the RSV minimal promoter (-51/+35) between
the BglII/HindIII sites of pGL2 (72). Synthetic
double-stranded oligonucleotides (Table 2
) corresponding to the -310/-278 region of the mouse
collagen X promoter or its mutants with a BamHI site (5')
and a BglII site (3') were ligated into the BglII
site of RSVLUC, creating (-310/-278)x1-RSVLUC, (-310/278)x2-RSVLUC,
(-310/-278)x3-RSVLUC, (-278/-310)x3-RSVLUC (reverse orientation),
(-310/-278)M1x3-RSVLUC, (-310/-278)M2x3-RSVLUC, and
(-310/-278)M3x3-RSVLUC. Three copies of the -327/-248 fragment
(described above) were also inserted into the KpnI site of
RSVLUC, creating (-327/-248)x3-RSVLUC. The copy number, orientation,
and sequence of inserts were confirmed by DNA sequencing. AP1x3-FIBLUC,
provided by Dr. S. J. OKeefe (Merck Research Laboratories,
Rahway, NJ), is a pGL2-based luciferase construct containing three
copies of the consensus AP-1 sequence (GTGACTCAGCGCGGA) upstream of the
human
-fibrinogen basal promoter. The TGFß-inducible p3TP-Lux
construct (27) was provided by Dr. Joan Massagué (Memorial
Sloan-Kettering Cancer Center, New York, NY). To generate an expression
plasmid for mouse c-fos (pcDNACFOS), the
EcoRI/SalI- digested fragment of mouse
c-fos cDNA (provided by Dr. Michael E. Greenberg, Harvard
Medical School, Boston, MA) was ligated into
EcoRI/XhoI-digested pcDNA3 expression vector
(Invitrogen, San Diego, CA).
Cell Culture and Transfection
C5.18 cells, derived from a fetal rat calvaria cell line
(20), were cultured in MEM-
containing 15% FBS. C5.18 cells were
transfected by the calcium phosphate-DNA coprecipitation method (73).
Briefly, C5.18 were plated on 12-well dishes (Costar, Cambridge, MA) at
1 x 105 cells per well. Forty eight hours later,
cells were incubated for 6 h with plasmids (2 µg/ml), which were
previously precipitated at 20 µg/ml in 25 mM HEPES (pH
7.1)/140 mM NaCl/0.75 mM
Na2HPO4/0.124 M CaCl2,
in growth medium. Cells were then treated for 2 min with 10%
dimethylsulfoxide (Hybrimax, Sigma) in PBS, rinsed twice with PBS, and
cultured in growth medium. C2C12 myoblasts (the American Type Culture
Collection; Rockville, MD), cultured in DMEM containing 10% FBS, were
transfected with plasmids (40 µg) by electroporation in Gene Pulser
Cuvettes (Bio-Rad Labs., Melville, NY) by using Gene Pulser (Bio-Rad
Labs.) at 960 µFarads/250 V. Twenty four hours after transfection,
cells were treated either with 100 ng/ml recombinant human OP-1 (2, 74), or 2 ng/ml porcine TGFß2 (R&D Systems, Minneapolis, MN), or 100
ng/ml human activin (provided by Dr. T. Matsumoto, University of Tokyo
School of Medicine, Tokyo, Japan) or 10 ng/ml human bFGF (R&D Systems)
for 24 h and then harvested for luciferase assays. Luciferase
assays were performed with a Berthold AutoCliniLumat Luminometer
(Bethold Analytical Instruments, Nashua, NH), by using the conditions
and buffers recommended by the Promega Luciferase Assay System. For
each experiment, transfections were performed in triplicate.
Transfection efficiencies were monitored with parallel transfections of
either the pGL2-Promoter Vector (SV40LUC; Promega) or RSVLUC, and the
results were standardized by calculating promoter activity relative to
that of SV40LUC or RSVLUC.
Gel Mobility Shift Assays
Nuclear extracts from cell lines were prepared by the method of
Dignam et al. (75) with the addition of 0.5 mM
4-(2-aminoethyl)-benzenesulfonylfluoride (Calbiochem, San Diego, CA), 5
µg/ml leupeptin, 5 µg/ml pepstatin, and 1 µg/ml aprotinin.
Nuclear extracts were dialyzed against buffer D containing 20%
glycerol, 20 mM HEPES (pH 7.9), 100 mM NaCl,
0.5 mM dithiothreitol, and 0.2 mM EDTA, then
stored in 20-µl aliquots at -70 C. For gel mobility shift assays,
100 pmol gel-purified double-stranded oligonucleotides were labeled
with T4 kinase and [
-32P]ATP (Amersham, Arlington
Heights, IL) or Klenow fragments and [
-32P]deoxy-CTP
(Amersham). Nuclear extracts (1020 µg) were incubated for 15 min at
20 C with approximately 0.05 pmol labeled double-stranded
oligonucleotides with 1 µg double-stranded
poly(deoxyinosinic-deoxycytidylic)acid·250
poly(deoxyino-sinic-deoxycytidylic)acid (Pharmacia, Piscataway, NJ)
and 10 µg BSA (Promega) in a total volume of 15 µl buffer D
containing 1 mM 4-(2-aminoethyl)-benzenesulfonylfluoride.
For antibody supershift assays, anti-human c-Fos polyclonal antibody
(Santa Cruz Biotechnology Inc., Santa Cruz, CA) was incubated with
reaction mixture for 15 min at 20 C before addition of an
oligonucleotide probe. Bound and free oligonucleotides were separated
on 420% acrylamide gels (Novex, San Diego, CA) in 0.375 x TBE
buffer (1 x TBE: 90 mM Tris base, 90 mM
boric acid, and 1.5 mM EDTA, pH 8.3) at 100 V. Gels were
dried and bands were visualized by autoradiography. Sequences of
oligonucleotides used for gel shift assays are described in Table 2
,
except for OCT, OCTA26 (76), and NP (48), previously described, and
E-box, which was purchased from Santa Cruz Biotechnology, Inc.
RNA Isolation and Northern Blot Analysis
Total cellular RNA was isolated by guanidine isothiocyanate and
phenol extraction as previously described (77). Total RNA (20 µg) was
electrophoresed through 1% agarose-formaldehyde (0.22 M)
and electroblotted to nylon filters (Hybond N; Amersham). The filters
were prehybridized in buffer containing 50% formamide, 5 x SSC
(1 x SSC = 0.15 M NaCl/0.015 M
sodium citrate), 5 x Denhardts solution, and sonicated salmon
sperm DNA (100 µg/ml) and hybridized at 42 C in fresh buffer
containing a mouse
-1 collagen X cDNA (provided by Dr. Benoit de
Crombrugghe, University of Texas, Houston, TX) or a human
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA probe labeled
with a random primer DNA-labeling kit (Pharmacia) and
[
-32P]deoxy-CTP (Amersham).
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. J. Massagué for the p3TP-Lux construct, Dr.
B. de Crombrugghe for mouse collagen X cDNA, Dr. M. E. Greenberg
for mouse c-fos cDNA, and Dr. S. J. OKeefe for the
AP-1
-fibrinogen promoter construct. We also thank "Laz-san" Dr.
Larry J. Suva, Dr. Alexs DAT-san, for critical reading of this
manuscript and helpful discussions; "Le-san" Dr. Le T. Duong and
Dr. Agi. E. Grigoriadis for helpful discussions; Mr. Rob. Vogel,
"Gre-yan" Mr. Gregg. Wesolowski, and Mr. Greg. Seedor for technical
support; and Mr. John Shockey and members of visual communication for
the art work. Special thanks to "Sev-san" Dr. Sevgi B. Rodan for
her contribution to this work.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Shun-ichi Harada, M. D., Department of Bone Biology and Osteoporosis Research, Merck Research Laboratories, West Point, Pennsylvania 19486.
Received for publication July 7, 1997.
Revision received August 18, 1997.
Accepted for publication August 20, 1997.
 |
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