Figure 1

Effects of TGF-β, BMP-2, and SB431542 on cultured MC3T3-E1 pre-osteoblasts. Cells were grown and treated with agents (50 ng/ml BMP-2, 5 ng/ml TGF-β1, and 1 μM or 5 μM SB431542) and subjected to a rapid staining protocol that visually indicated osteogenic differentiation at 3 days (A, Alkaline Phosphatase) or matrix mineralization at 10 days (B, Alizarin Red S). These results confirmed that SB431542 can promote both early osteoblast differentiation and mature osteoblast function in vitro, and these results were independently repeated. To confirm mechanistically the effects of these compounds on downstream signaling (SMAD activity), Western analysis was performed on day 3 samples using pSMAD1, pSMAD2, SMAD1, SMAD2 and α-Tubulin antibodies. These data indicated that BMP-2 and SB431542 could promote SMAD1 phosphorylation and reduce SMAD2 phosphorylation.