FIG. 7.

p-SMAD1/5/8 nuclear translocation requires cell spreading and ROCK signaling. (A) Immunofluorescence images of serum-starved hMSCs plated on fibronectin stamped islands and treated with or without BMP-2 (100 ng/mL) for 1 h. Green, stress fiber; red, p-SMAD1/5/8; blue, DAPI. (B) Quantification of ratio of nuclear translocated p-SMAD1/5/8 in hMSCs plated on different sizes of fibronectin-stamped area. (C) Immunofluorescence images of Y27632 (10 μM) or blebbstatin (25 μM) pretreated hMSCs followed by BMP treatment for 1 h. Green, stress fiber; red, p-SMAD1/5/8; blue, DAPI. (D) Quantification of BMP-2-induced nuclear translocated p-SMAD1/5/8 in the presence of Y27632 or blebbstatin. *P<0.05 versus paired control; ^#P<0.05 versus BMP-treatment at 10,000 μm² and BMP treat alone. Scale bar, 20 μm. (E) Model of cell shape regulates BMP-2-induced osteogenic differentiation. Cell shape acts as a mechanical cue that cooperates with BMP to induced osteogenesis. Rho/ROCK-mediated cell tension is not only required but also induced in response to BMP-2 treatment. The RhoA/ROCK signaling regulates nuclear translocation of p-SMAD, which is responsible for BMP-2 induced osteogenesis.