Figure 7.

Enhanced osteoblast mineralization by EDA-containing fibronectin is mediated by α4β1 integrin. A, the CS1 peptide, which binds α4β1, enhances osteoblast mineralization after 2–3 weeks in mineralizing medium. CS1 and scrambled peptide were added at 25 μg/ml with every medium change (biological replicates: n = 7/9 in 3 experiments). B, deletion of β1 integrin in osteoblasts using the osterix promoter attached to Cre in mice homozygous for floxed β1 integrin decreases β1 expression as shown by immunoblotting (biological replicates: n = 10/6 in 3 experiments). Primary osteoblasts were evaluated 3 days after isolation. C, whereas transfection of wild-type osteoblasts with FN-EDA enhances differentiation, FN-EDA fails to increase mineralization in the absence of β1 integrin. However, deletion of β1 integrin itself suppresses mineralization considerably (biological replicates: n = 8/9/6/6 in 3 experiments). Osteoblasts were transfected and cultured in mineralizing medium for 2–3 weeks. D, chemical inhibition of α4β1 prevents enhancement of mineralization by FN-EDA but not by FN-EDB (biological replicates: n = 6/6/6/9/10/6 in 3 experiments). Osteoblasts were cultured in mineralizing medium for 2–3 weeks, and the inhibitor was added with each medium change (3 times/week) at 1.8 nm. Results are expressed as mean ± S.D. (error bars). Bars, 1 mm. *, p < 0.05; **, p < 0.01; ***, p < 0.005.