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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}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 {alpha}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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 (30–250 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 (30–250 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. 1Go and Table 1Go). 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. 1Go). 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. 8Go).



View larger version (33K):
[in this window]
[in a new window]
 
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.

 

View this table:
[in this window]
[in a new window]
 
Table 1. OP-1 Dose-Dependent Up-Regulation of Collagen X Promoter Activity in C5.18 Cells

 


View larger version (33K):
[in this window]
[in a new window]
 
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.

 
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. 2AGo and 2BGo). A further 26-bp deletion (-301/-248) abolished the OP-1 response (Fig. 2AGo). 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. 2BGo). 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.



View larger version (31K):
[in this window]
[in a new window]
 
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. 2AGo and %RSVLUC in Fig. 2BGo) ±SD.

 
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. 3AGo). Nor did TGFß2 or activin increase collagen X mRNA levels in C5.18 cells (Fig. 3BGo). 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.



View larger version (56K):
[in this window]
[in a new window]
 
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)x2–90COLXLUC 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.

 
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 {gamma}-fibrinogen minimal promoter (AP1x3-FIBLUC) (Fig. 4Go). Neither OP-1 nor bFGF had an effect on the minimal {gamma}-fibrinogen promoter (data not shown). Furthermore, a mouse c-fos expression plasmid cotransfected with (-327/-248)x2–90COLXLUC increased its expression by 2-fold, and OP-1 caused a further 2-fold stimulation (Fig. 5Go). 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.



View larger version (42K):
[in this window]
[in a new window]
 
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.

 


View larger version (27K):
[in this window]
[in a new window]
 
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)x2–90COLXLUC, 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.

 
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. 6Go). 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. 6AGo, 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. 6AGo, 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. 6AGo, 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. 6AGo; lanes 6–8), was absent after the additional mutation in the A/T-rich sequence (M4) (Fig. 6AGo, lanes 9–11). 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. 6BGo). This supershift was blocked by the antigenic peptide, suggesting the involvement of Fos family proteins in this complex.



View larger version (34K):
[in this window]
[in a new window]
 
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 1–5), COLX(-310/-278) with a mutation in the AP-1 like sequence (M1; lanes 6–8) or COLX(-310/-278) with mutations in the AP-1-like sequence and in the A/T-rich sequence (M4; lanes 9–11). 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 1–5) 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).

 
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. 6CGo). 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. 6CGo).

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. 6CGo). These findings suggest that C5.18 nuclear proteins interact with the A/T-rich sequence (Fig. 6CGo, 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. 6Go, A and B; band C) and 2) via the A/T-rich MEF-2-like sequence (Fig. 6Go, 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. 7Go, lanes 6–9), consistent with the mutational analysis (Fig. 4Go). 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. 7Go, lanes 1–5). This band is competed by cold homologous oligonucleotide. OP-1 treatment had no effect on band D (Fig. 7Go, lanes 1–5), 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. 7Go, band B).



View larger version (74K):
[in this window]
[in a new window]
 
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 1–5) or with COLX(-310/-278)M2 mutated in the A/T- rich sequence (lanes 6–9). Competition experiments contained 400-fold molar excess of homologous oligonucleotide (lane 5).

 
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. 8Go). The -314/-295 probe formed specific slowly migrating complexes (Fig. 9AGo, 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. 9AGo, lane 2, and Fig. 7Go, 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. 9BGo, bands E). These results suggest that the slowly migrating bands E are formed by the endogenous MEF-2 proteins present in C2C12 myoblasts.



View larger version (24K):
[in this window]
[in a new window]
 
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 3–6) 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 3–6) 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).

 
The MEF-2 oligonucleotide probe also formed complexes with C5.18 nuclear extracts (Fig. 9CGo, bands B). These are similar to the fast migrating complexes formed with the collagen X A/T-rich sequence (Fig. 6CGo and Fig. 7Go, 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. 9CGo, 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.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 8Go).

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 {alpha}-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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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)x2–90COLXLUC or (-310/-248)x2–90COLXLUC, 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 2Go) 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. O’Keefe (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 {gamma}-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).


View this table:
[in this window]
[in a new window]
 
Table 2. Oligonucleotides Used in This Study

 
Cell Culture and Transfection
C5.18 cells, derived from a fetal rat calvaria cell line (20), were cultured in MEM-{alpha} 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 [{gamma}-32P]ATP (Amersham, Arlington Heights, IL) or Klenow fragments and [{alpha}-32P]deoxy-CTP (Amersham). Nuclear extracts (10–20 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 4–20% 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 2Go, 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 Denhardt’s solution, and sonicated salmon sperm DNA (100 µg/ml) and hybridized at 42 C in fresh buffer containing a mouse {alpha}-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 [{alpha}-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. O’Keefe for the AP-1 {gamma}-fibrinogen promoter construct. We also thank "Laz-san" Dr. Larry J. Suva, Dr. Alex’s 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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Kingsley DM 1994 The TGF-beta superfamily: new members, new receptors, and new genetic tests of function in different organisms. Genes Dev 8:133–146[CrossRef][Medline]
  2. Sampath TK, Maliakal JC, Hauschka PV, Jones WK, Sasak H, Tucker RF, White K, Coughlin JE, Tucker MM, Pang RHL, Corbett C, Özkaynak E, Opperman H, Rueger DC 1992 Recombinant human osteogenic protein (OP-1) induces new bone formation in vivo with specific activity comparable with natural bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro. J Biol Chem 267:20352–20362[Abstract/Free Full Text]
  3. Lyons KM, Hogan BLM, Robertson EJ 1995 Colocalization of BMP-7 and BMP-2 RNAs suggests that these factors cooperatively mediate tissue interactions during murine development. Mech Dev 50:71–83[CrossRef][Medline]
  4. Dudley AT, Lyons KM, Robertson EJ 1995 A requirement for bone morphogenetic protein-7 during development of the mammalian kidney and eye. Genes Dev 9:2795–2807[Abstract]
  5. Luo G, Hofmann C, Bronckers A, Sohocki M, Bradley A, Karsenty G 1995 BMP-7 is an inducer of nephrogenesis, and is also required for eye development and skeletal patterning. Genes Dev 9:2808–2820[Abstract]
  6. Dudley AT, Robertson EJ 1997 Overlapping expression domains of bone morphogenetic protein family members potentially account for limited tissue defects in BMP7 deficient embryos. Dev Dyn 208:349–362[CrossRef][Medline]
  7. Cancedda R, Cancedda FD, Castagnola P 1995 Chondrocyte differentiation. Int Rev Cytol 159:265–358[Medline]
  8. Reddi AH 1995 Cartilage morphogenesis: role of bone and cartilage morphogenetic proteins, homeobox genes and extracellular matrix. Matrix Biol 14:599–606[Medline]
  9. Lu Valle P, Ninomiya Y, Rosenblum ND, Olsen BR 1988 The type X collagen gene: intron sequences split the 5'-untranslated region and separate the coding regions for the non-collagenous amino-terminal and triple-helical domains. J Biol Chem 263:18378–18385[Abstract/Free Full Text]
  10. Elima K, Eerola I, Rosati R, Metsäranta M, Garofalo S, Perälä M, de Crombrugghe B, Vuorio E 1993 The mouse collagen X gene: complete nucleotide sequence, exon structure, and expression pattern. Biochem J 289:247–253[Medline]
  11. Thomas JT, Cresswell CJ, Rash B, Nicolai H, Jones H, Solomon E, Grant ME, Boot-Handford RP 1991 The human collagen X gene: complete primary translated sequence and chromosomal localization. Biochem J 280:617–623[Medline]
  12. Thomas JT, Sweetman WA, Cresswell CJ, Wallis GA, Grant ME, Boothandford RP 1995 Sequence comparison of 3 mammalian type-X collagen promoters and preliminary functional analysis of the human promoter. Gene 160:291–296[CrossRef][Medline]
  13. Jacenko O, Lu Valle P, Olsen BR 1993 Spondylo-metaphyseal dysplasia in mice carrying a dominant negative mutation in a matrix protein specific for cartilage-to-bone transition. Nature 365:56–61[CrossRef][Medline]
  14. Lu Valle P, Iwamoto M, Fanning P, Pacifici M, Olsen BR 1993 Multiple negative elements in a gene that codes for an extracellular matrix protein, collagen X, restrict expression to hypertrophic chondrocytes. J Cell Biol 121:1173–1179[Abstract]
  15. Long FX, Linsenmayer TF 1995 Tissue specific regulation of the Type X collagen gene: analyses by in vivo footprinting and transfection with a proximal promoter region. J Biol Chem 270:31310–31314[Abstract/Free Full Text]
  16. Yamaguchi A, Katagiri T, Ikeda T, Wozney JM, Rosen V, Wang EA, Kahn AJ, Suda T, Yoshiki S 1991 Recombinant human bone morphogenetic protein-2 stimulates osteoblastic maturation and inhibits myogenic differentiation in vitro. J Cell Biol 113:681–687[Abstract]
  17. Rosen V, Nove J, Song JJ, Thies RS, Cox K, Wozney JM 1994 Responsiveness of clonal limb bud cell lines to bone morphogenetic protein 2 reveals a sequential relationship between cartilage and bone cell phenotype. J Bone Miner Res 9:1759–1768[Medline]
  18. Asahina I, Sampath TK, Nishimura I, Hauschka PV 1993 Human osteogenic protein-1 induces both chondroblastic and osteoblastic differentiation of osteoprogenitor cells derived from new born rat calvaria. J Cell Biol 123:921–933[Abstract]
  19. Katagiri T, Yamaguchi A, Komaki M, Abe E, Takahashi N, Ikeda T, Rosen V, Wozney JM, Fujisawa-Sehara A, Suda T 1994 Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage. J Cell Biol 127:1755–1766[Abstract]
  20. Grigoriadis AE, Heersche JNM, Aubin JE 1996 Analysis of chondroprogenitor frequency and cartilage differentiation in a novel family of rat clonal chondrogenic cell lines. Differentiation 60:299–307[CrossRef][Medline]
  21. Grigoriadis AE, Heersche JNM, Aubin JE 1988 Differentiation of muscle, fat, cartilage, and bone from progenitor cells present in a bone-derived clonal cell population: effect of dexamethasone. J Cell Biol 106:2139–2151[Abstract]
  22. Harada S, Machwate M, Sampath TK, Nagy JA, Brown LF, Fisher JE, Rodan GA, Rodan SB 1995 Induction of vascular endothelial growth factor by osteogenic protein-1 in vitro and in vivo. J Bone Miner Res 10:S421 (Abstract)
  23. Perides G, Safran RM, Downing LA, Charness ME 1994 Regulation of neural adhesion molecule and L1 by the transforming growth factor-beta superfamily: selective effects of the bone morphogenetic proteins. J Biol Chem 269:765–770[Abstract/Free Full Text]
  24. Tavella S, Raffo P, Tacchetti C, Cancedda R, Castagnola P 1994 N-CAM and N-cadherin expression during in vitro chondrogenesis. Exp Cell Res 215:354–362[CrossRef][Medline]
  25. Oberlender SA, Tuan RS 1994 Expression and functional involvement of N-cadherin in embryonic limb chondrogenesis. Development 120:177–187[Abstract/Free Full Text]
  26. Vainio S, Karavanova I, Jowett A, Thesleff I 1993 Identification of BMP-4 as a signal mediating secondary interactions between epithelial and mesenchymal tissues during early tooth development. Cell 75:45–58[Medline]
  27. Wrana JL, Attisano L, Cárcamo J, Zentella A, Doody J, Laiho M, Wang X-F, Massagué J 1992 TGF beta signals through a heteromeric protein kinase receptor complex. Cell 71:1003–1014[Medline]
  28. Olson EN, Perry M, Schulz RA 1995 Regulation of muscle differentiation by the MEF2 family of MADS box transcription factors. Dev Biol 172:2–14[CrossRef][Medline]
  29. Grigoriadis AE, Wang ZQ, Wagner EF 1995 Fos and bone cell development: lessons from a nuclear oncogene. Trends Genet 11:436–441[CrossRef][Medline]
  30. Dony C, Gruss P 1987 Proto-oncogene c-fos expression in growth regions of fetal bone and mesodermal web tissue. Nature 328:711–714[CrossRef][Medline]
  31. Sandberg M, Vuorio T, Hirvonen H, Alitalo K, Vuorio E 1988 Enhanced expression of TGF-beta and c-fos mRNA in the growth plates of developing human long bones. Development 102:461–470[Abstract]
  32. Inaoka T, Lean JM, Bessho T, Chow JWM, Mackay A, Kokubo T, Chambers TJ 1995 Sequential analysis of gene expression after an osteogenic stimulus: c-fos expression is induced in osteocytes. Biochem Biophys Res Commun 217:264–270[CrossRef][Medline]
  33. Ohta S, Yamamura K, Lee H, Okumura H, Kasai R, Hiraki Y, Ikeda T, Iwasaki R, Kikuchi H, Konishi J, Shigeno C 1991 Fracture healing induces expression of the proto-oncogene c-fos in vivo: possible involvement of the Fos protein in osteoblastic differentiation. FEBS Lett 284:42–45[CrossRef][Medline]
  34. Wroblewski J, Edwall-Arvidsson C 1995 Inhibitory effects of basic fibroblast growth factor on chondrocyte differentiation. J Bone Miner Res 10:735–742[Medline]
  35. Ohta S, Hiraki U, Shigeno C, Suzuki F, Kasai R, Ikeda T, Kohno H, Lee K, Kikuchi H, Konishi J, Bentz H, Rosen DM, Yamamuro T 1992 Bone morphogenetic proteins (BMP-2 and BMP-3) induce the late phase expression of the proto-oncogene c-fos in murine osteoblastic MC3T3–E1 cells. FEBS Lett 314:356–360[CrossRef][Medline]
  36. Chang E, Goldberg H 1995 Requirements for transforming growth factor-beta regulation of the pro-alpha-2(I) collagen and plasminogen activator inhibitor-1 promoters. J Biol Chem 270:4473–4477[Abstract/Free Full Text]
  37. Chung KY, Agarwal A, Uitto J, Mauviel A 1996 An AP-1 binding sequence is essential for regulation of the hu-man alpha-2(I) collagen (Col1{alpha}2) promoter activity by transforming growth factor-beta. J Biol Chem 271:3272–3278[Abstract/Free Full Text]
  38. Datto MB, Yu Y, Wang XF 1995 Functional analysis of the transforming growth factor-beta responsive elements in the Waf1/Cip1/P21 promoter. J Biol Chem 270:28623–28628[Abstract/Free Full Text]
  39. Kerr RD, Miller DB, Matrisian LM 1990 TGF-beta1 inhibition of transin/stromelysin gene expression is mediated through a Fos binding sequence. Cell 61:267–278[Medline]
  40. Määtä A, Glumoff V, Paakkonen P, Liska D, Penttinen RPK, Elima K, 1993 Nuclear factor binding to an AP-1 site is associated with the activation of pro-alpha 1(I)-collagen gene in dedifferentiating chondrocytes. Biochem J 294:365–371[Medline]
  41. Schule R, Umesone K, Mangelsdorf DJ, Bolado J, Pike JW, Evans RM 1990 Jun-Fos and receptors for vitamins A and D recognize a common response element in the human osteocalcin gene. Cell 61: 497–504
  42. Goto K, Rosen V, Wozney JM, Kronenberg HM, Demay MB 1994 Sequences that mediated the induction of osteocalcin gen transcription by rhBMP-2. Bone Miner Res 9:S254 (Abstract)
  43. Xu RH, Dong ZG, Maeno M, Kim J, Suzuki A, Ueno N, Sredni D, Colburn NH, Kung HF 1996 Involvement of ras/raf/Ap-1 in BMP-4 signaling during Xenopus embryonic development. Proc Natl Acad Sci USA 93:834–838[Abstract/Free Full Text]
  44. Goswami S, Qasba P, Ghatpande S, Carleton S, Deshpande AK, Baig M, Siddiqui MAQ 1994 Differential expression of the myocyte enhancer factor 2 family of transcription factors in development: the cardiac factor BBF-1 is an early marker for cardiogenesis. Mol Cell Biol 14:5130–5138[Abstract]
  45. Molkentin JD, Markham BE 1994 An M-CAT binding factor and an RSRF-related A-rich binding factor positively regulate expression of the alpha-cardiac myosin heavy-chain gene in vivo. Mol Cell Biol 14:5056–5065[Abstract]
  46. Grayson J, Williams RS, Yu Y-T, Bassel-Duby R 1995 Synergistic interaction between heterologous upstream activation elements and specific TATA sequences in a muscle specific promoter. Mol Cell Biol 15:1870–1878[Abstract]
  47. Zhu H, Nguyen VTB, Brown AB, Pourhosseini A, Garcia AV, Vanbilsen M, Chien KR 1993 A novel, tissue restricted zinc-finger protein (HF-1b) binds to the cardiac regulatory element (HF-1b/MEF-2) In the rat myosin light-chain 2 gene. Mol Cell Biol 13:4432–4444[Abstract]
  48. Komuro I, Schalling M, Jahn L, Bodmer R, Jenkins NA, Copeland NG, Izumo S 1993 Gtx: a novel murine homeobox-containing gene, expressed specifically in glial cells of the brain and germ cells of testis, has a transcriptional repressor activity in vitro for a serum-inducible promoter. EMBO J 12:1387–1401[Abstract]
  49. Cserjesi P, Lilly B, Hinkley C, Perry M, Olson EN 1994 Homeodomain protein MHox and MADS protein myocyte enhancer-binding factor-2 converge on a common element in the muscle creatine kinase enhancer. J Biol Chem 269:16740–16745[Abstract/Free Full Text]
  50. Massagué J 1996 TGF beta signaling: receptors, transducers, and Mad proteins. Cell 85:947–950[Medline]
  51. Molkentin JD, Li L, Olson EN 1996 Phosphorylation of the MADS-box transcription factor MEF2C enhances its DNA binding activity. J Biol Chem 271:17199–17204[Abstract/Free Full Text]
  52. Han J, Jiang Y, Li Z, Kravchenko VV, Ulevitch RJ 1997 Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation. Science 386:296–299[CrossRef]
  53. Treisman R 1994 Ternary complex factors: growth factor regulated transcriptional activators. Curr Opin Genet Dev 4:86–101
  54. Molkentin JD, Black BL, Martin JF, Olson EN 1995 Cooperative activation of muscle gene expression by MEF2 and myogenic bHLH proteins. Cell 83:1125–1136[Medline]
  55. Oue Y, Kanatani H, Kiyoki M, Eto Y, Ogata E, Matsumoto T 1994 Effect of local injection of activin A on bone formation in newborn rats. Bone 15:361–366[Medline]
  56. Centrella M, Horovitz MC, Wozney JM, McCarthy TL 1994 Transforming growth factor-beta gene family members and bone. Endocr Rev 15:27–39[Medline]
  57. Noda M, Camilliere JJ 1989 In vivo stimulation of bone formation by transforming growth factor-beta. Endocrinology 124:2991–2994[Abstract]
  58. Luyten FP, Chen P, Paralkar V, Reddi AH 1994 Recombinant bone morphogenetic protein-4, transforming growth factor-beta 1, and activin A enhance the cartilage phenotype of articular chondrocytes in vitro. Exp Cell Res 210:224–229[CrossRef][Medline]
  59. Ballock TR, Heydemann A, Wakefield LM, Flanders KC, Roberts AB, Sporn MB 1993 TGF beta 1 prevents hypertrophy of epiphyseal chondrocytes: regulation of gene expression for cartilage matrix proteins and metalloproteases. Dev Biol 158:414–429[CrossRef][Medline]
  60. Chen P, Vukicevic S, Sampath TK, Luyten FP 1995 Osteogenic protein-1 promotes growth and maturation of chick sternal chondrocytes in serum-free cultures. J Cell Sci 108:105–114[Abstract/Free Full Text]
  61. Inagaki Y, Truter S, Ramirez F 1994 Transforming growth factor beta stimulates alpha-2(I) collagen gene expression through a cis-acting element that contains an Sp1 binding site. J Biol Chem 269:14828–14834[Abstract/Free Full Text]
  62. Riccio A, Pedone PV, Lund LR, Olesen T, Olsen HS, Andreasen PA 1992 Transforming growth factor beta1-responsive element: closely associated binding sites for USF and CCAAT-binding transcription factor-nuclear factor in the type 1 plasminogen activator inhibitor gene. Mol Cell Biol 12:1846–1855[Abstract]
  63. Li JM, Nichols MA, Chandrasekharan S, Xiong Y, Wang XF 1995 Transforming growth factor beta activates the promoter of cyclin-dependent kinase inhibitor P15Ink4B through an Sp1 consensus site. J Biol Chem 270:26750–26753[Abstract/Free Full Text]
  64. Tamura M, Noda M 1994 Identification of a DNA sequence involved in osteoblast-specific gene expression via interaction with helix-loop-helix (HLH)-type transcription factors. J Cell Biol 126:773–782[Abstract]
  65. Raftery LA, Twombly V, Wharton K, Gelvart WM 1995 Genetic screens to identify elements of the decapentaplegic signaling pathway in Drosophila. Genetics 139:241–254[Abstract/Free Full Text]
  66. Wrana JL, Attisano L 1996 MAD-related proteins in TGF-ß signaling. Trends Genet 12:493–497[CrossRef][Medline]
  67. Hoodless PA, Haerry T, Abdollah S, Stapleton M, O’Connor MB, Attisano L, Wrana JL 1996 MADR1, a MAD-related protein that functions in BMP2 signaling pathways. Cell 85:489–500[Medline]
  68. Kretzschmar M, Liu F, Hata A, Doody J, Massagué J, Barbacid M 1997 The TGF-ß family mediator smad1 is phosphorylated directly and activated functionally by the BMP receptor kinase. Genes Dev 11:984–995[Abstract]
  69. Liu F, Hata A, Baker JC, Doody J, Carcamo J, Harland RM, Massagué J 1996 A human Mad protein acting as a BMP-regulated transcriptional activator. Nature 381:620–623[CrossRef][Medline]
  70. Chen X, Rubock MJ, Whitman M 1996 A transcriptional partner for MAD proteins in TGF-ß signalling. Nature 383:691–696[CrossRef][Medline]
  71. Kim J, Johnson K, Chen HJ, Carroll S, Laughon A 1997 Drosophila Mad binds to DNA and directly mediates activation of vestigial by Decapentaplegic. Nature 388:304–308[CrossRef][Medline]
  72. Towler DA, Rodan GA 1995 Identification of a rat osteocalcin promoter 3',5'-cyclic adenosine monophosphate response region containing two PuGGTCA steroid hormone receptor binding motifs. Endocrinology 136:1089–1096[Abstract]
  73. Chen C, Okayama H 1988 Calcium phosphate-mediated gene transfer: a highly efficient system for stably transforming cells with plasmid DNA. Biotechniques 6:632–638[Medline]
  74. Jones WK, Richmond EA, White K, Sasak H, Kusmik W, Smart J, Oppermann H, Rueger DC, Tucker RF 1994 Osteogenic protein-1 (OP-1) expression and processing in Chinese hamster ovary cells: isolation of a soluble complex containing the mature and pro-domains of OP-1. Growth Factors 11:215–225[Medline]
  75. Dignam J, Lebowitz R, Roeder R 1983 Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11:1475–1489[Abstract]
  76. Towler DA, Bennett CD, Rodan GA 1994 Activity of the rat osteocalcin basal promoter in osteoblastic cells is dependent upon homeodomain and CP1 binding motifs. Mol Endocrinol 8:614–624[Abstract]
  77. Chomoczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[CrossRef][Medline]