JBJS | Intimate Relationship Between TGF-β/BMP Signaling and Runt Domain Transcription Factor, PEBP2/CBF

The Journal of Bone & Joint Surgery, Volume 83, Issue 1 suppl 1

Scientific Article | March 01, 2001

Intimate Relationship Between TGF-β/BMP Signaling and Runt Domain Transcription Factor, PEBP2/CBF

Suk-Chul Bae; Kycong-Sook Lee; Yu-Wen Zhang; Yoshiaki Ito

Investigation performed at the Department of Biochemistry, School of Medicine, and Medical Research Institute, Chungbuk National University, Cheongju, Korea; and the Department of Viral Oncology, Institute for Virus Research, Kyoto University, Kyoto, Japan
Suk-Chul Bae Kycong-Sook Lee Department of Biochemistry, School of Medicine, Chungbuk National University, Cheongju, Korea 361-763
Yoshiaki Ito Yu-Wen Zhang Department of Viral Oncology, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan. E-mail address for Yoshiaki Ito: yito@virus.kyoto-u.ac.jp
In support of their research or preparation of this manuscript, one or more of the authors received grants or outside funding from Basic Research Program of the Korea Science and Engineering Foundation and Grant-in-Aid 09253220 for Priority Area in Cancer Research from the Ministry of Education, Science, Sports and Culture, Japan. None of the authors received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.

The Journal of Bone & Joint Surgery. 2001; 83:S48-S55

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Abstract

Background: When C2C12 pluripotent mesenchymal precursor cells are treated with transforming growth factor-ß1 (TGF-ß1), terminal differentiation into myotubes is blocked. Treatment with bone morphogenetic protein-2 (BMP-2) not only blocks myogenic differentiation but also induces osteoblastic differentiation. However, the molecular mechanisms governing the ability of TGF-ß and BMP to induce ligand-specific responses and inhibit myogenic differentiation are not known. The objective of our studies was to elucidate the molecular mechanisms that block myoblastic differentiation and induce osteoblastic differentiation in C2C12 cells.

Methods: Induction of RUNX2/PEBP2aA/Cbfa1 by TGF-ß and BMP was examined by electrophoretic mobility shift assay (EMSA) and Northern blot analysis. C2C12 cells stably expressing RUNX2 or Smad, or both, were established, and the role of these genes in the process of osteoblastic differentiation was analyzed by examining the expression of osteoblast-specific markers.

Results: Treatment of C2C12 with TGF-ß and BMP-induced RUNX2/PEBP2aA/Cbfa1, a global regulator of osteogenesis. Cooperation between RUNX2 and BMP-activated Smad induced osteoblastic differentiation.

Conclusions: Both TGF-ß and BMP activate transcription of RUNX2, which is sufficient to inhibit myogenesis. To induce osteogenesis, BMP-induced RUNX2 must cooperate with BMP-activated Smads.

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Introduction

Introduction | Materials and Methods | Results and Discussion | References

The pluripotent mesenchymal precursor cell line C2C12 undergoes differentiation to form multinucleated myotubes in response to low mitogen concentration in culture medium. Transforming growth factor-ß1 (TGF-ß1) inhibits the myotube formation of C2C12 cells without inducing osteoblastic phenotypes. On the other hand, bone morphogenetic proteins (BMPs) not only inhibit myotube formation of C2C12 cells but also induce osteoblastic differentiation. Therefore, C2C12 cells are useful tools to study the ligand-specific signaling mechanisms of TGF-ß and BMPs ¹ . The recent identification of the gene encoding the a subunit (RUNX2/PEBP2aA/Cbfa1) of the transcription factor PEBP2/CBF ² as a bone-specific gene is a significant milestone in osteoblast biology ^3-5 . Intramembranous and endochondral ossification is blocked in RUNX2 knockout mice by an arrest in osteoblastic maturation ^4,5 . The RUNX2 gene has been mapped to human chromosome 6p21 ⁶ , which was also found to be the genetic locus for cleidocranial dysplasia (CCD) syndrome ⁷ . CCD is an autosomal-dominant human bone disease characterized by hypoplastic clavicles, patent fontanelles and sutures, and other multiple skeletal disorders. Recently, a heterozygous mutation of the RUNX2 gene was found in several patients with CCD ^7-9 . These observations suggest that the disease is caused by heteroinsufficiency of RUNX2. However, the molecular mechanism underlying the pathogenesis of CCD in patients with heterozygous mutations in the RUNX2 gene is poorly understood. In this study, we investigated the molecular mechanisms that block myoblastic differentiation and induce osteoblastic differentiation in C2C12 cells.

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Fig. 1-A:: Fig. 1 Identification of transforming growth factor-ß (TGF-ß) responsive element (TßRE) binding protein by electrophoretic mobility shift assay (EMSA). Fig. 1-A Oligonucleotide sequence of the TßRE identified in the Ig Ca promoter. ¹¹ and three mutant DNAs-M1 (one mismatch in the putative RUNX binding site [CACCACA]), M2 (a perfect match [GACCACA]), and M3 (putative Smad binding site [CAGACA]), respectively-are shown. Putative RUNX2 and Smad binding sites are indicated by underlining and a dotted line, respectively, and mutations are marked by asterisks.

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Fig. 1-B:: Fig. 1 Identification of transforming growth factor-ß (TGF-ß) responsive element (TßRE) binding protein by electrophoretic mobility shift assay (EMSA). Fig. 1-B EMSA was performed by using the TßRE probe and nuclear lysates obtained from C2C12 cells untreated or treated with TGF-ß1 or bone morphogenetic protein-2 (BMP-2) for 24 hours. A 50-fold molar excess of unlabeled mutant oligonucleotide was incubated with the nuclear lysates as competitor DNA. The arrow and arrowhead indicate the TßRE binding complex and free probe, respectively.

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Fig. 1-C:: Fig. 1 Identification of transforming growth factor-ß (TGF-ß) responsive element (TßRE) binding protein by electrophoretic mobility shift assay (EMSA). Fig. 1-C EMSA was performed with use of the same nuclear lysates and TßRE probes in the presence or absence of polyclonal antibody, which recognized all members of the Runx a subunit (anti-a; lanes 2 and 5) or b subunit (anti-ß; lanes 3 and 6), or monoclonal antibody, which specifically recognized RUNX2 (anti-RUNX2; lanes 8, 10, and 12). The arrowhead and arrows indicate the positions of RUNX2 and RUNX-antibody complexes, respectively. (Reprinted, with permission, from Lee KS, Him HJ, Li QL, Chi XZ, Ueta C, Komori T, Wozney JM, Kim EG, Choi JY, Ryoo HM, Bae SC. Runx2/PEBP2aA/Cfa1 is a common target of TGF-ß1 and BMP-2 and cooperation between Runx2 and Smad5 induces osteoblast specific gene expression in the pluripotent mesenchymal precursor cell line C2C12. Mol Cell Biol 2000;20,8783.)

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Fig. 2-A:: Fig. 2 Time course of the induction of RUNX2. Fig. 2-A Induction of RUNX2 expression was analyzed by electrophoretic mobility shift assay (EMSA) using nuclear lysates prepared from cells treated with transforming growth factor-ß1 (TGF-ß1) for 0, 4, 12, 18, 24, 72, and 120 hours. EMSA was performed using TGF-ß responsive element (TßRE) probe in the presence or absence of a 50-fold molar excess of unlabeled M3 as competitor. The arrow indicates the TßRE binding complex. Ct = competitor.

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Fig. 2-B:: Fig. 2 Time course of the induction of RUNX2. Fig. 2-B C2C12 cells were treated with TGF-ß1 (5 ng/ml) or bone morphogenetic protein-2 (BMP-2) (300 ng/ml) for the indicated times, and total RNA was prepared. Northern blotting was performed using the C-terminal coding region of RUNX2 as a probe (Rx2-Ct). A probe prepared from the GAPDH coding sequence was used as a loading control. (Reprinted, with permission, from Lee KS, Him HJ, Li QL, Chi XZ, Ueta C, Komori T, Wozney JM, Kim EG, Choi JY, Ryoo HM, Bae SC. Runx2/PEBP2aA/Cbfa1 is a common target of TGF-ß1 and BMP-2 and cooperation between Runx2 and Smad5 induces osteoblast specific gene expression in the pluripotent mesenchymal precursor cell line C2C12. Mol Cell Biol . 2000;20:8783.)

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Fig. 3:: Pattern of gene expression following bone morphogenetic protein-2 (BMP-2) and transforming growth factor-ß1 (TGF-ß1) treatment of control C2C12 and C2C12-Rx2 cells. Control C2C12 cells (lanes 1-3) and C2C12-Rx2 cells stably expressing RUNX2 (lanes 4-6) were treated with TGF-ß1 (5 ng/ml) or BMP-2 (300 ng/ml) for 3 days. Total RNA was extracted and analyzed by Northern blotting with probes homologous to osteocalcin (OC), collagen type I (Col-I), fibronectin (FN), or MyoD. (Reprinted, with permission, from Lee KS, Him HJ, Li QL, Chi XZ, Ueta C, Komori T, Wozney JM, Kim EG, Choi JY, Ryoo HM, Bae SC. Runx2/PEBP2aA/Cbfa1 is a common target of TGF-ß1 and BMP-2 and cooperation between Runx2 and Smad5 induces osteoblast specific gene expression in the pluripotent mesenchymal precursor cell line C2C12. Mol Cell Biol . 2000;20:8783.)

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Fig. 4:: Fig. 4 Myotube formation of C2C12 cells stably expressing RUNX2 and CCDaA376. Fig. 4-A Western blot showing expression of RUNX2 and CCDaA376 (lane 1, mock; lane 2, RUNX2; lane 3, CCDaA376). (Reprinted, with permission, from Zhang YW, Yasui N, Ito K, Huang G, Fuji M, Hanai J, Nogami H, Ochi T, Miyazono K, Ito Y. A RUNX2/PEBP2alpha A/CBFA1 mutation displaying impaired transactivation and Smad interaction in cleidocranial dysplasia. Proc Natl Acad Sci U S A . 2000;97:10549.) Fig. 4-B Myotube formation stained with anti-troponin T antibody in C2C12 cells transfected with vector alone. Fig. 4-C C2C12 cells stably expressing RUNX2, stained with anti-troponin T antibody. Fig. 4-D C2C12 cells stably expressing CCDaA376, stained with anti-troponin T antibody. Cells were cultured in Dulbecco's modified Eagle medium containing 2.5% fetal bovine serum for 5 days.

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Fig. 5-A:: Fig. 5 Failure of CCDaA376 to interact with Smads. Fig. 5-A Schematic illustration of RUNX2 (shown as aA), its truncated constructs, and the CCDaA376 mutant. The amino acid sequence comparison between activation domains of PEBP2aA/RUNX2 and PEBP2aB/RUNX1 and the position of the premature stop codon in CCDaA376 (marked by an asterisk) also are shown. NLS = nuclear localization signal.

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Figs. 5-B and 5-C:: Fig. 5 Failure of CCDaA376 to interact with Smads. Fig. 5-B The physical interaction between RUNX2 and Smad3 as examined by GST-pull down assay ²⁰ . Fig. 5-CIn vivo interaction. COS-7 cells were cotransfected with expression plasmids coding for FLAG-tagged Smad-3 (for lanes 1-6) or FLAG-tagged Smad1 (for lanes 7 and 8), a constitutively active form of type I transforming growth factor-ß (TßR-I; for lanes 1-6) or bone morphogenetic protein (BMPR-IA; for lanes 7 and 8) receptor, and RUNX2 mutants, the structures of which are shown in A . RUNX2 proteins were immunoprecipitated from cell lysates with anti-FLAG antibody followed by immunoblotting (WB) using anti-aA8G5 antibody. In the lower panel, the co-immunoprecipitation of RUNX2 and aA(1-424) with Smads is indicated. Expression levels of individual proteins are shown in the upper and middle panels. (Reprinted, with permission, from Zhang YW, Yasui N, Ito K, Huang G, Fuji M, Hanai J, Nogami H, Ochi T, Miyazono K, Ito Y. A RUNX2/PEBP2alpha A/CBFA1 mutation displaying impaired transactivation and Smad interaction in cleidocranial dysplasia. Proc Natl Acad Sci U S A . 2000;97:10549.)

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Fig. 6:: Impaired responsiveness of CCDaA376 to transforming growth factor-ß (TGF-ß) signaling. P19 cells were co-transfected with the reporter plasmid of Ca (WT), Ca (TßRE-mS), Ca (TßRE-mP), or Ca (mSP) (0.2 g) and the expression plasmids for RUNX2 (shown as aA) or CCDaA376 (0.1 µg) and Smad3/4 (0.1 µg each) with or without TßR-I (0.1 µg). Relative luciferase activities are shown as fold induction. The expression plasmids for FLAG-tagged Smad1, Smad3, Smad4, and TßR-I and BMPR-IA have been described previously ²¹ . The luciferase reporter plasmids containing the germline immunoglobulin Ca promoter, Ca(WT), and its mutant forms, and the expression plasmid for GST-Smad3 were described elsewhere ¹⁷ . (Reprinted, with permission, from Zhang YW, Yasui N, Ito K, Huang G, Fuji M, Hanai J, Nogami H, Ochi T, Miyazono K, Ito Y. RUNX2/PEBP2alpha A/CBFA1 mutation displaying impaired transactivation and Smad interaction in cleidocranial dysplasia. Proc Natl Acad Sci U S A . 2000;97:10549.)

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Fig. 7-A:: Fig. 7 Induction of alkaline phosphatase (ALP) by cooperation between RUNX2 and Smad5. Fig. 7-A C2C12 (control), C2C12-Rx2, C2C12-Sm5, and C2C12-Sm5-Rx2 cells were cultured in the absence of bone morphogenetic protein-2 (BMP-2). Total RNA was prepared, and the levels of ALP mRNA were analyzed by Northern blot hybridization.

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Fig. 7-B:: Fig. 7 Induction of alkaline phosphatase (ALP) by cooperation between RUNX2 and Smad5. Fig. 7-B The same cells were treated with the indicated concentration of BMP-2 for 3 days, and ALP enzyme activities were assayed as described by Katagiri et al ¹ .

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Fig. 7-C:: Fig. 7 Induction of alkaline phosphatase (ALP) by cooperation between RUNX2 and Smad5. Fig. 7-C ALP enzyme activities of C2C12 and CCD-C2C12 in response to BMP-2 were analyzed. (Reprinted, with permission, from Lee KS, Him HJ, Li QL, Chi XZ, Ueta C, Komori T, Wozney JM, Kim EG, Choi JY, Ryoo HM, Bae SC. Runx2/PEBP2aA/Cbfa1 is a common target of TGF-ß1 and BMP-2 and cooperation between Runx2 and Smad5 induces osteoblast specific gene expression in the pluripotent mesenchymal precursor cell line C2C12. Mol Cell Biol . 2000;20:8783.)

Materials and Methods

Introduction | Materials and Methods | Results and Discussion | References

Materials

Recombinant human BMP-2 (rhBMP-2) was a kind gift of Dr. John M. Wozney (Genetics Institute, Cambridge, Massachusetts). Recombinant human BMP-7 (rhBMP-7, rhOP-1) was a gift from T. K. Sampath (Creative Biomolecules, Hopkinton, Massachusetts). Recombinant human TGF-ß1 (rhTGF-ß1) and anti-FLAG M2 antibody were from Sigma Chemical (St. Louis, Missouri). Anti-Smad1 and anti-HA antibodies were purchased from Santa Cruz.

Plasmids

The human RUNX2 cDNA encoding the entire region from exon 1 to exon 7 was generated by combining the partial cDNA we isolated previously6 with reverse transcriptase-polymerase chain reaction (RT-PCR) fragments amplified from exon 1 and exon 6. The mammalian expression plasmid pEF-aA was constructed by inserting the PEBP2aA cDNA into the BstXI site of pEF-Bos. pEF-CCDaA376, pEF-aA(1-424), pEF-aA(1-388), pEF-aA(1-340), pEF-aA(1-280), pEF-aA(1-221), and pEF-aA(1-214) were constructed by replacing the KpnI-BamHl fragment of pEF-aA with PCR-amplified fragments containing an artificial termination codon at the end of the amino acid residues indicated in parentheses.

Electrophoretic Mobility Shift Assay (EMSA)

Nuclear protein extracts were prepared from cells stimulated with rhTGF-ß1 or rhBMP-2 as described previously ¹⁰ . Protein concentrations of the extracts were determined with use of the Bradford assay (Bio-Rad, Richmond, California). A double-stranded DNA probe, the TGF-ß responsive element of the Ig Ca promoter ¹¹ (TßRE), was prepared and used for EMSA as described previously ¹² . The oligonucleotide sequences used in EMSAs are given in Fig. 1-A .

Immunoprecipitation (IP) and Western Blotting

COS-7 cells were transiently transfected with expression plasmids by FuGENE 6. Cell extracts were prepared 48 hours after transfection ¹³ . IP and Western blotting were performed as described ¹³ . Anti-aA8G5 monoclonal antibody was raised against an Escherichia coli -produced recombinant PEBP2aA protein.

Results and Discussion

Introduction | Materials and Methods | Results and Discussion | References

RUNX2 is Induced by TGF-ß1 and BMP-2

With use of the TßRE present in the immunoglobulin Ca (Ig Ca) promoter ¹¹ ( Fig. 1-A ), the DNA-ßinding activity of the TßRE binding protein was examined by EMSA. TßRE binding activity was detected in C2C12 cells and was significantly increased by TGF-ß1 and BMP-2 ( Fig. 1-B , compare lanes 1, 6, and 11). The addition of M1 or M3 mutant competitors effectively competed out the entire TßRE-protein interaction, whereas the M2 mutant competitor did not ( Fig. 1-B ). This suggests that the induced protein bound to the second RUNX consensus sequence but not to the first (the M1 site), which is imperfect ( Fig. 1-A ). To examine whether the TGF-ß1 or BMP-2-induced TßRE binding protein is actually a RUNX protein, EMSA supershift assays were performed with RUNX2 or PEBP2ß/Cbfb-specific antibodies. As shown in Fig. 1-C , the TßRE binding protein-DNA complex was supershifted by a polyclonal antibody that recognizes RUNX1, 2, and 3 (lanes 2 and 5) and by a monoclonal antibody that recognizes RUNX2 (lanes 8, 10, and 12), as well as by PEBP2ß/Cbfb-specific anti-serum (lanes 3 and 6), whereas no supershifted band was observed with use of pre-immune serum (lanes 1 and 4). These results indicate that, in C2C12 cells, the major TßRE binding protein induced by TGF-ß1 and BMP-2 is RUNX2. A time-course study showed that the induction of the RUNX2 protein and mRNA reached a maximum as early as 4 and 2 hours after TGF-ß1 stimulation, respectively, and gradually decreased thereafter even though the cells were continuously stimulated by TGF-ß1 ( Fig. 2-A , Fig. 2-B ).

RUNX2 Mediates the Common Activities of TGF-ß1 and BMP-2

Since TGF-ß1 and BMP-2 induce RUNX2, experiments were carried out to determine whether exogenous expression of RUNX2 could mimic the common effect of TGF-ß1 and BMP-2. For this purpose, C2C12 cells stably expressing RUNX2 (C2C12-Rx2) were obtained, and molecular markers for TGF-ß1 and BMP-2 stimulation were analyzed. TGF-ß1 and BMP-2 are known to suppress MyoD ¹ and induce collagen a1(I) and fibronectin gene expression ¹⁴ . Up or downregulation of these marker genes by TGF-ß1 and BMP-2 was confirmed in control C2C12 cells ( Fig. 3 , lanes 1-3). Constitutively expressed RUNX2 also suppressed MyoD and induced collagen a1(I) and fibronectin gene expression in C2C12 cells ( Fig. 3 , lane 4). The induction of these major components of the extracellular matrix and the suppression of myoD expression, which is critically important for myogenic differentiation ¹⁵ , by RUNX2 suggest that RUNX2 mediates the common activities of TGF-ß1 and BMP-2.

Conversely, osteocalcin, a mineralized tissue-specific gene ¹⁶ induced by BMP-2 but not by TGF-ß1, was not up-regulated by RUNX2 expression alone ( Fig. 3 , lane 4).

However, in the presence of BMP-2, overexpression of RUNX2 led to a significantly increased induction of osteocalcin over the level of osteocalcin in the control of BMP-2-treated cells (compare lanes 3 and 6). Although this result suggests that RUNX2 also has an important role to play in osteoblastic differentiation, other factors in addition to RUNX2 appear to be necessary.

The exogenous expression of RUNX2 suppresses MyoD expression; therefore, we examined whether it could also block myogenic differentiation. C2C12 cells expressing RUNX2 (C2C12-Rx2) and C-terminally truncated RUNX2 mutant CCDaA376 (CCD-C2C12) were obtained ( Fig. 4-A ). CCDaA376, which lacks the carboxy-terminal of 130 amino acids9, was isolated from a Japanese patient with CCD. Myotube formation was completely blocked in the cells expressing RUNX2, as well as in the TGF-ß1 or BMP-2-treated cells, indicating that RUNX2 by itself is sufficient to block myogenic differentiation ( Fig. 4-C ). In contrast, exogenous expression of CCDaA376 did not block myogenic differentiation ( Fig. 4-D ), suggesting that the C-terminal region of RUNX2 missing in CCDaA376 is required to block myogenesis.

RUNX2, but not CCDaA376, Cooperatively Functions with Smad on the TGF-ß Responsive Element

RUNX2, as well as RUNX1 and RUNX3, physically associates with Smad1, 2, 3, and 5 in vivo¹⁷ . To establish the physiological effect of the association between RUNX2 and Smads, we used the CCDaA376 mutant. In GST-pull down assays ( Fig. 5-B ), the interaction of RUNX2 with Smad3 was significantly reduced when CCDaA376 was employed. The interaction of RUNX2 with Smad3 was also examined by immunoprecipitation followed by Western blotting with similar results - that is, the interaction became undetectable when aA(1-388) or shorter constructs were used ( Fig. 5-B ). Similarly, the mutant CCDaA376 hardly interacted with Smad3 in vivo ( Fig. 5-C ). Essentially the same result was obtained with Smad1 ( Fig. 5-C ), Smad2, and Smad5 (data not shown).

The functional relationship between PEBP2 and Smads has been successfully studied using the germline immunoglobulin Ca promoter, since the TßRE consists only of PEBP2 and Smad binding sites ¹⁷ . The Ca promoter was cooperatively stimulated by RUNX2 and Smads in a TGF-ß-dependent manner ( Fig. 6 ). However, CCDaA376 displayed greatly reduced responsiveness to TGF-ß, which is in agreement with the impaired interaction between CCDaA376 and Smads ( Fig. 5-A , Figs. 5-B and 5-C ). The result established that RUNX2 has the capacity to functionally cooperate with Smads and that CCDaA376 is severely impaired in this ability.

Cooperation Between RUNX2 and BMP-Activated Smad Induces Osteoblast-Specific Gene Expression

Induction of alkaline phosphatase (ALP) activity by BMP is considered to be an indicator of an early stage of osteoblastic differentiation ¹ . TGF-ß1, which can block myogenic differentiation of C2C12 cells but cannot induce osteoblastic differentiation, does not induce ALP activity. Consistent with the differential effect of BMP and TGF-ß, BMP-specific Smads, but not TGF-ß-specific Smads, have been shown to be responsible for the induction of ALP activity in C2C12 cells ¹⁸ . It has also been reported that exogenous expression of RUNX2 can induce ALP activity in C3H10T1/2 cells ¹⁹ . We confirmed the induction of ALP activity by overexpressing RUNX2 in C2C12 cells ( Fig. 7-A , lanes 1 and 2; Fig. 7-B ). However, the level of ALP activity induced by RUNX2 in the absence of exogenous BMP-2 was very low compared with that in C2C12 cells treated with 300 ng/ml BMP-2 alone ( Fig. 7-B ). These observations together with the result shown in Fig. 3 indicate that RUNX2 alone is not sufficient for the induction of osteoblast-specific gene expression and that an additional BMP-2-specific signal is required. Signal-specific Smad proteins physically interact with RUNX2; therefore, we surmised that BMP-specific Smad proteins might be additionally required for the induction of osteoblast-specific gene expression. To prove this, we obtained C2C12 cells stably expressing both Smad5 and RUNX2 (C2C12-Sm5-Rx2) and examined the level of ALP mRNA with use of Northern blot analysis ( Fig. 7-A ). We confirmed the low level of ALP mRNA in C2C12-Sm5 and C2C12-Rx2 cells in the absence of BMP-2 stimulation ( Fig. 7-A , lanes 2 and 3). Remarkably, ALP expression was strongly enhanced in C2C12-Sm5-Rx2 cells ( Fig. 7-A , lane 4). We further analyzed ALP expression in these cells by treating them with various concentrations of BMP-2 and measuring the ALP activity. C2C12-Sm5-Rx2 cells showed quite strong ALP activity even in the absence of BMP-2 ( Fig. 7-B ). The activity was higher than that of cells overexpressing either Smad5 or RUNX2 alone. Exogenous expression of CCDaA376, which is unable to interact with Smads, did not induce a detectable level of ALP activity ( Fig. 7-C ). Even after subsequent BMP treatment, the induction of ALP activity was severely restricted ( Fig. 7-C ). The results suggest that RUNX2 and Smads cooperate to induce osteoblast cells and that they are critical elements in an elaborate molecular switch that triggers differentiation into the osteoblastic lineage after BMP treatment. The pathological basis of the CCD syndrome may be due, at least in part, to haploinsufficiency of the responsiveness of RUNX2 to TGF-ß superfamily signaling.

In this study, we identified RUNX2 as the common and major target of TGF-ß1 and BMP-2 in C2C12 cells. RUNX2 was found to be responsible for the common activities of TGF-ß and BMP, suppression of the expression of the myogenic master gene, MyoD, and induction of components of the extracellular matrix but insufficient for osteoblast-specific gene expression. Osteoblast-specific gene expression was mediated by cooperation between BMP-activated Smad and BMP-induced RUNX2. Furthermore, the truncated mutant of RUNX2, CCDaA376, originally identified in a patient with CCD, failed to cooperate with Smads and was unable to induce osteoblast-specific gene expression in C2C12 on stimulation by BMP-2. Therefore, cooperation between RUNX2 and Smads is essential for the TGF-ß/BMP signaling in C2C12 differentiation, and the pathogenesis of CCD may be related to the defect in responsiveness of RUNX2 to TGF-ß superfamily signaling during bone formation. These observations provide important insights into how the downstream actors of the TGF-ß1 and BMP-2 signaling pathways mediate mesenchymal precursor cell differentiation. Induction of RUNX2 is essential for the common activities of TGF-ß and BMP, and cooperation between RUNX2 and receptor-activated Smads (R-Smads) could mediate ligand-specific gene expression.

Note: This work was supported by Grant No. 1999-2-20900-008-5 from the Basic Research Program of the Korea Science and Engineering Foundation to S.C.B. and Grant-in-Aid 09253220 for Priority Area in Cancer Research from the Ministry of Education, Science, Sports and Culture, Japan, to Y.I.

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Introduction | Materials and Methods | Results and Discussion | References

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Suk-Chul Bae, Kycong-Sook Lee, Yu-Wen Zhang, Yoshiaki Ito; Intimate Relationship Between TGF-β/BMP Signaling and Runt Domain Transcription Factor, PEBP2/CBF. The Journal of Bone & Joint Surgery. 2001 Mar;83(1_suppl_1):S48-S55.

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