Figure 5.

Conditional deletion of Rac1 in preosteoblasts interferes with integrin signaling. (A) Only transfection with fibronectin containing EDA (FN-EDA) increases Rac1 mRNA and protein expression. n = 8/7/7 and 10/9/6. Rac1 mRNA was adjusted to HPRT and Rac1 protein to GAPDH. (B) Transfection with FN-EDA enhances mineralization in CT Rac1^fl/fl and in Osx Rac1 osteoblasts. Unlike CT Rac1^fl/fl cells, however, alkaline phosphatase in the media and osteocalcin mRNA do not respond in Osx Rac1. Newborn calvarial osteoblasts were isolated, transfected with the FN or FN-EDA construct and cultured for 2–3 weeks in mineralizing conditions in the presence of vitamin C, β-glycerophosphate, and dexamethasone added fresh with each medium change (3 times/week). At the end, the wells were stained using von Kossa stain and the media evaluated for alkaline phosphatase. Osteocalcin/HPRT mRNA was evaluated in sister wells that were not stained or in separate experiments and corrected to the housekeeping gene HPRT. Mice were not exposed to doxycycline at any time. n = 7/6/5/7, 6/5/5/4, 10/3/8/3. (C) ERK and AKT phosphorylation increased upon transfection with FN-EDA in CT Rac1^fl/fl cells. In Osx Rac1 the phosphorylation of both increased with FN and did not increase further with FN-EDA. Cells were transfected and left two days before collecting the cell lysate for Western blotting. n = 7/6/9/12, 7/7/5/7. ANOVA was performed and whenever significant, the values were compared between every two groups in all subpanels using t-tests.