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. 2000 Dec;20(23):8783-92.
doi: 10.1128/MCB.20.23.8783-8792.2000.

Runx2 is a common target of transforming growth factor beta1 and bone morphogenetic protein 2, and cooperation between Runx2 and Smad5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12

K S Lee  1 , H J Kim, Q L Li, X Z Chi, C Ueta, T Komori, J M Wozney, E G Kim, J Y Choi, H M Ryoo, S C Bae
Affiliations

Runx2 is a common target of transforming growth factor beta1 and bone morphogenetic protein 2, and cooperation between Runx2 and Smad5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12

K S Lee et al. Mol Cell Biol. 2000 Dec.

Abstract

When C2C12 pluripotent mesenchymal precursor cells are treated with transforming growth factor beta1 (TGF-beta1), terminal differentiation into myotubes is blocked. Treatment with bone morphogenetic protein 2 (BMP-2) not only blocks myogenic differentiation of C2C12 cells but also induces osteoblast differentiation. The molecular mechanisms governing the ability of TGF-beta1 and BMP-2 to both induce ligand-specific responses and inhibit myogenic differentiation are not known. We identified Runx2/PEBP2alphaA/Cbfa1, a global regulator of osteogenesis, as a major TGF-beta1-responsive element binding protein induced by TGF-beta1 and BMP-2 in C2C12 cells. Consistent with the observation that Runx2 can be induced by either TGF-beta1 or BMP-2, the exogenous expression of Runx2 mediated some of the effects of TGF-beta1 and BMP-2 but not osteoblast-specific gene expression. Runx2 mimicked common effects of TGF-beta1 and BMP-2 by inducing expression of matrix gene products (for example, collagen and fibronectin), suppressing MyoD expression, and inhibiting myotube formation of C2C12 cells. For osteoblast differentiation, an additional effector, BMP-specific Smad protein, was required. Our results indicate that Runx2 is a major target gene shared by TGF-beta and BMP signaling pathways and that the coordinated action of Runx2 and BMP-activated Smads leads to the induction of osteoblast-specific gene expression in C2C12 cells.

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Figures

FIG. 1
FIG. 1
Identification of the TβRE binding protein by EMSA. (A) Oligonucleotide sequences of the TβRE identified in the Ig Cα promoter (29) 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]). Putative Runx2 and Smad binding sites are indicated by solid and dotted underlining, respectively, and mutated nucleotides are marked by asterisks. (B) EMSA was performed by using the TβRE probe and nuclear lysates obtained from C2C12 cells untreated or treated with TGF-β1 or BMP-2 for 24 h. The reactions were performed in the presence or absence of PEBP2β/Cbfβ protein. A 50-fold molar excess of unlabeled mutant oligonucleotide was incubated with the nuclear lysates as competitor DNA (Ct). The arrow and arrowhead indicate the TβRE binding complex and free probe, respectively. (C) EMSA was performed by using the same nuclear lysates and TβRE probes in the presence or absence of a polyclonal antibody which recognized all members of the Runx α subunit (anti-α; lane 2 and 5) or β subunit (anti-β; lane 3 and 6) or a monoclonal antibody which specifically recognized Runx2 (anti-Runx2; lanes 8, 10, and 12). The arrowheads and arrows indicate the positions of Runx2 and Runx-antibody complexes, respectively. (D) Time course induction of Runx2 was analyzed by EMSA using nuclear lysates prepared from cells treated with TGF-β1 for 0, 4, 12, 18, 24, 72, and 120 h. A nuclear extract prepared from cells cultured for 72 h in the absence of TGF-β1 was used as a control. EMSA was performed with the TβRE probe in the presence or absence of a 50-fold molar excess of unlabeled M3 as competitor. The arrow indicates the TβRE binding complex. Ct, competitor.
FIG. 2
FIG. 2
Induction of Runx2 mRNA by TGF-β1 and BMP2. (A) C2C12 cells were treated with TGF-β1 (5 ng/ml) or BMP-2 (300 ng/ml) for the indicated times, and total RNA was prepared. Northern blotting was performed using PEBP2αA cDNA (38), which contains the common region of Runx2-αA1 and Runx2-til-1, as a probe for Runx2 (Rx2-Ct). (B) Total RNAs were prepared from C2C12 cells treated with BMP-2 (300 ng/ml) for 6 h and analyzed by Northern blot hybridization using the Rx2-Ct probe. The same blot was stripped and rehybridized with Runx2-αA1 (38)- or Runx2-til-1 (48)-specific probes. A probe prepared from the GAPDH coding sequence was used as a loading control.
FIG. 3
FIG. 3
Binding of Runx2 to the Runx binding site is essential for the TGF-β1 responsiveness of the TβRE. (A) Diagrams of luciferase reporter constructs. Two reporters for the TβRE of the Ig Cα promoter were constructed using the pGL3-promoter plasmid (Promega) containing the simian virus 40 promoter (SV40 Pr) as a backbone, i.e., one with the wild-type (WT) TβRE (pGL3-TβRE) DNA and the other with the TβRE mutated at the Runx binding site (pGL3-M2). Two copies of the element were inserted for each plasmid construct. (B) pGL3, pGL3-M2, and pGL3-TβRE reporters were transfected into C2C12 cells with the Runx2 expression plasmid (Runx2) or the constitutively active TGF-β receptor I expression plasmid (TβR-I) or with both. Cells were harvested 48 h after transfection, and luciferase activities were assayed. (C and D) The same reporters were transfected into MC3T3-E1 and H1-127-30 cells with or without the Runx2 expression plasmid (Runx2) and cultured in the presence or absence of TGF-β1 for 24 h. Luciferase activities were measured, and relative activities are shown.
FIG. 4
FIG. 4
Morphological changes of C2C12 cells stably expressing Runx2. (A) Western blotting showing overexpression of Runx2 in C2C12-Rx2 cells. Cytoplasmic (C) and nuclear (N) protein extracts were prepared from C2C12 and C2C12-Rx2 cells, and Runx2 protein was detected by Western blotting. An arrow indicates Runx2 protein. (B) C2C12 cells containing the empty vector were cultured for 10 days in differentiation medium (5% FBS in DMEM) in the presence or absence of TGF-β1 (5 ng/ml) or BMP-2 (300 ng/ml). C2C12-Rx2 cells were cultured under the same conditions in the absence of TGF-β1 and BMP-2. After incubation, morphological changes were compared.
FIG. 5
FIG. 5
Pattern of gene expression following BMP-2 and TGF-β1 treatment of C2C12 and C2C12-Rx2 cells. Control C2C12 (lane 1 to 3) and C2C12-Rx2 (lane 4 to 6) cells were treated with TGF-β1 (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.
FIG. 6
FIG. 6
Suppression of Runx2 expression during myogenic differentiation. C2C12 cells were cultured in DMEM containing 5% FBS for 1 day (lane 1) or 7 days (lane 2). Total RNA was prepared, and the levels of myoD and Runx2 mRNA were analyzed by Northern blotting.
FIG. 7
FIG. 7
Induction of ALP by cooperation between Runx2 and Smad5. (A) Western blot showing exogenous expression of Smad5 in C2C12-Sm5 and C2C12-Sm5-Rx2 cells. The arrow indicates Smad5 protein. (B) C2C12 (control), C2C12-Rx2, C2C12-Sm5, and C2C12-Sm5-Rx2 cells were cultured in the absence of BMP-2. Total RNA was prepared, and the levels of ALP mRNA were analyzed by Northern blot hybridization. (C) 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. (20).
FIG. 8
FIG. 8
Involvement of Smads in the induction of Runx2 expression. (A) Control C2C12 cells and C2C12-Sm5 cells were treated with the indicated concentration of BMP-2 for 6 h. Total RNA was prepared from the cells, and the Runx2 mRNA level was analyzed by Northern blotting using Rx2-Ct as a probe. (B) Protein lysates were obtained from control C2C12 (lane 1) and C2C12-Sm5 (lane 2) cells, and the level of Runx2 protein was analyzed by Western blotting. (C) C2C12 cells were cultured in the absence or presence of BMP-2 (300 ng/ml) and cycloheximide (CHX; 5 μg/ml) as indicated for 6 h. Total RNAs were prepared, and the Runx2 mRNA level was analyzed by Northern blot hybridization using the Rx2-Ct probe.
FIG. 9
FIG. 9
A model for the common and distinct activities of TGF-β and BMP in C2C12 cells. Runx2 is an indirect target of the Smad signaling pathway. Induction of Runx2 is essential for the common activities of TGF-β and BMP. Ligand-specific gene expression, however, requires cooperation between Runx2 and receptor-activated Smads (R-Smad).
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