C2C12 cells were treated with different concentrations of BMP-2 (50, 100, and 200 ng/ml) or transfected with different amounts of Runx2 or Smurf1 expression plasmids (0.2, 0.4, and 0.8 µg/dish, 6-mm culture dish). Total RNA and cell lysates were extracted 24 h after transfection or BMP-2 treatment. Changes in Smad6 mRNA and protein levels were analyzed by real-time PCR (a) and Western blotting (b). BMP-2 and Runx2 stimulated and Smurf1 inhibited Smad6 mRNA and protein expression in a dose-dependent manner.

a, diagram shows the construct harboring wild-type (wt) or mutant (mt) OSE2-a and OSE2-b sites derived from −1191/+45 reporter. The wt and mt sequences of OSE2-a and OSE2-b sites are indicated. b, C2C12 cells were transfected with wt and mt −1191/+45 Smad6 reporter with Runx2 expression plasmid or treated with BMP-2 (100 ng/ml). Cell lysates were collected 24 h after transfection, and a luciferase assay was performed. Mutation of OSE2-a caused a significant reduction in basal promoter activity and completely abolished BMP-2- or Runx2-induced promoter activity of the −1191/+45 reporter. Mutation of OSE2-b had no significant effect on the basal promoter activity or the responsiveness to Runx2 or BMP-2. *, p < 0.05, unpaired Student’s t test, compared with vector alone. c, C2C12 cells were transfected with wt and mt −2006/+45 Smad6 reporter with Runx2 or Smad1 expression plasmid or treated with BMP-2 (100 ng/ml). Mutation of OSE2-a in −2006/+45 reporter significantly reduced basal promoter activity and inhibited BMP-2, Runx2, or Smad1-induced promoter activity. *, p < 0.05, unpaired Student’s t test, compared with vector alone.

a, diagram shows the structure of the mouse Smad6 gene. The putative Runx2 binding site (OSE2-a) and priming sites for ChIP assay are indicated. b, IP was performed using anti-Runx2 antibody (lanes 1, 3, and 4) or anti-FLAG control antibody (lane 2). The PCR was performed using primer set A (lanes 1, 2, and 5) to detect Runx2 binding and the PCR product (171 bp) of OSE2-a region (−1108/−937, lane 1) was amplified using the samples of cross-linked IP products as templates. Primer set B (−1968/−1839, lanes 3 and 6) and set C (+281/+432, lanes 4 and 7) were used as negative controls for PCR. Results showed that Runx2 binds specifically to the OSE2-a region (lane 1). c, C2C12 cells were transfected with Myc-Smad1 plasmid and treated with or without BMP-2 for 2 h before the ChIP assay. The IP was performed using the anti-Myc (lanes 1, 3, and 4) antibody and the anti-FLAG control antibody (lane 2). The PCR was performed using primer set A (lanes 1, 2, and 5) for detecting Smad1 interaction with the OSE2-a region (−1108/−937) and primer set B (−1968/−1839) and C (+281/+432) as negative controls. Results showed that Smad1 interacts with the OSE2-a region (lane 1).

In the absence of BMP-2, Smurf1 binds Runx2 and induces Runx2 degradation. In the presence of BMP-2, Smad1 replaces Smurf1 and binds to the OSE2 site.

a, diagram shows the structure of the mouse Smad6 promoter −2006/+45 construct. The putative Runx2 binding sites (OSE2-a, OSE2-b), and Smad1/5 binding region (three overlapping GC-rich sequences) are indicated. b, C2C12 cells were co-transfected with the Smad6 promoter construct (−2006/+45) or three deletion constructs (−1191/+45, −829/+45, and −374/+45) with Runx2 or Smad1 expression plasmid or treated with BMP-2 (100 ng/ml). Cell lysates were collected 24 h after transfection, and a luciferase assay was performed. BMP-2 and Runx2 stimulated the promoter activity of −2006/+45 and −1191/+45 constructs with similar potency but had no effect on −829/+45 and −374/+45 reporters. Transfection of Smad1 further enhanced Runx2-induced promoter activity of −2006/+45 and −1191/+45 constructs compared with the transfection of each plasmid separately. *, p < 0.05, unpaired Student’s t test, compared with vector transfection alone.

a, nuclear extracts from C2C12 cells were assayed by gel shift using ³²P end-labeled oligonucleotide probes derived from the mouse Smad6 promoter (−1046/−1026). Lane 1, free probe control (without nuclear extracts); lane 2, addition of nuclear extracts only. The binding of OSE2-a was competed with excess amounts of cold wild-type (lanes 3 and 4) or mutant (lanes 5 and 6) oligonucleotide (1:10 and 1:100 ratio). The same nuclear extracts were also examined in supershift assays using antibody for Runx2 (lane 7) or IgG control antibody (lane 8). DNA protein binding complexes and the Runx2-specific supershifted band were observed (lane 7). b, binding of Runx2, Smad1, or Smurf1 was further determined by gel shift assay using in vitro translated GST-Runx2, GST-Smad1, and GST-Smurf1 proteins. Smad1 or Smurf1 did not bind the OSE2-a site (lanes 3 and 5). The binding of Runx2 was reduced in the presence of Smurf1 (lane 4).

a, Smad6 promoter −2006/+45 and −1191/+45 were co-transfected with different amounts of Smurf1 (0.2, 0.4, and 0.8µg/dish, 6-mm culture dish) or mSmurf1 (0.8 µg/dish) expression plasmids into C2C12 cells. Cell lysates were collected 24 h after transfection, and a luciferase assay was performed. Smurf1 but not mSmurf1 inhibited the promoter activity of −2006/+45 and −1191/+45 constructs in a dose-dependent manner. *, p < 0.05, one-way analysis of variance followed by Dunnett’s test, compared with vector alone. b, Smurf1 ChIP assay. C2C12 cells were transfected with Smad1 and treated with BMP-2 (100 ng/ml). Cell lysates were collected 2 h after BMP-2 treatment. The IP was performed using the anti-Smurf1 (lanes 1, 3, and 4) or anti-FLAG (lane 2, negative control) antibodies. The PCR was performed using primer set A (lanes 1, 2, and 5) (OSE2-a region, −1108/−937), B (−1968/−1839), and C (+281/+432) (negative control). Results showed that Smurf1 interacts with the OSE2-a region in the absence of BMP-2 and Smad1 (upper panel). With BMP-2 treatment and Smad1 transfection, Smurf1 was no longer able to interact with the OSE2-a region of the Smad6 promoter. c, qChIP assay. Copy numbers of the DNA fragment containing OSE2-a site (−1108/−937) in anti-Runx2 and anti-Smurf1 immunoprecipitated samples before (Input DNA) and after IP were quantified by real-time PCR using primer set A. The bars indicate final values of the percentage of the DNA input obtained from specific antibody-immunoprecipitated samples subtracting the percentage of DNA input obtained from anti-FLAG control antibody-immunoprecipitated samples. The results showed that BMP-2/Smad1 enhanced Runx2 binding and inhibited Smurf1 binding to the OSE2-a site. *, p < 0.05, (n = 4), unpaired Student’s t test, Runx2 binding was compared with untreated group. **, p < 0.05 (n=4), unpaired Student’s t test, Smurf1 binding was compared with untreated group.