Fig. 5.

Inhibition of osteoblast proliferation and differentiation in Smad1^Col1a1 mice. (A and B) Paraffin-embedded sections of calvariae were stained with BrdU. The BrdU-positive osteoblasts were indicated by red arrows and a 22% reduction of BrdU-positive osteoblasts was found in Smad1^Col1a1 mice. Unpaired Student’s t-test, P = 0.002 (n = 4). (C) Primary osteoblasts isolated from Smad1^Col1a1 mice and littermate control mice were cultured for 10 days in the absence or presence of BMP-2 (100 ng/ml). von Kossa staining was performed after the cell culture was ended and the formation of mineralized bone nodules was analyzed. In osteoblasts derived from Smad1^Col1a1 mice, the formation of mineralized bone nodules was inhibited. (D) Primary osteoblasts isolated from Smad1^Col1a1 mice and littermate control mice were transfected with BMP signaling reporter construct 12× SBE-OC-Luc and treated with 4, 20 and 100 ng/ml of BMP-2. The luciferase activity was measured and normalized to β-gal activity. In osteoblasts derived from Smad1 cKO mice, BMP signaling was significantly inhibited compared with cells isolated from littermate control mice. One-way ANOVA followed by Dunnett’s test, BMP-2 (4 ng/ml), P = 0.003; BMP-2 (20 ng/ml), P = 0.014; BMP-2 (100 ng/ml), P < 0.001 (n = 3) between control and Smad1^Col1a1 cells.