Figure 9.

EDB-containing fibronectin requires a functional RGD sequence to enhance osteoblast differentiation. A, in control cells (β3 CT), transfection with either FN-EDA or FN-EDB increased mineralization, whereas transfection with FN-EDB in β3 knock-out (β3 KO) osteoblasts no longer increased mineralization. FN-EDA transfected in β3 knock-out cells still enhanced mineralization. Osteoblasts were isolated from β3 CT and β3 KO mice; transfected with the FN, FN-EDA, or FN-EDB constructs; cultured for 2–3 weeks in mineralizing medium; and evaluated (biological replicates: n = 8/8/7/8/7/7 in 2 experiments). B, in the absence of β3 integrin, FN-EDB transfection fails to increase FAK phosphorylation (biological replicates: n = 10/10/5/8 in 4 experiments). Osteoblasts were transfected and cultured for 2 days in medium containing fibronectin-depleted FCS, and cell lysates were collected. C, echistatin, which binds to αvβ3, enhances osteoblast differentiation in vitro (biological replicates: n = 4/4 in 2 experiments). Echistatin was added at 0.27 nm with every medium change to osteoblasts cultured in mineralization medium. Wells were evaluated after 2–3 weeks. D, echistatin stimulates integrin signaling, as shown for FAK phosphorylation (biological replicates: n = 9/9 in 3 experiments). Echistatin was added at 0.27 nm to osteoblasts exposed overnight to FCS-free medium, and cell lysates were collected 15 min later. E, transfection of FN-EDB containing the mutated, non-functional RGD sequence (FN-EDB RGE) fails to enhance osteoblast differentiation compared with cells expressing the non-mutated FN-EDB construct containing functional RGD (biological replicates evaluated by von Kossa staining: n = 9/12/11 in 4 experiments). Osteoblasts were transfected with FN, FN-EDB, or FN-EDB RGE. Cells were cultured for 2–3 weeks in mineralizing medium, and wells were evaluated after von Kossa staining. Results are expressed as mean ± S.D. (error bars). Bars, 1 mm. *, p < 0.05; **, p < 0.01; ***, p < 0.005.