BIO activates β-catenin signaling stimulates osteoblast
differentiation and bone formation. Primary calvarial osteoblasts were
isolated 3-day-old WT neonatal mice and cultured with or without BIO (1 μM)
for different periods of time as indicated. The β-catenin protein levels
and nuclear translocation were examined by western blot and immunostaining.
BIO increased protein levels of active form of β-catenin and reached its
maximal effect at 4 hours (A). BIO also induced β-catenin nuclear
translocation (B). Primary calvarial osteoblasts, isolated from TOPGal
transgenic mice, were treated with BIO (1 μM) for 24 hours and β-Gal
activity was measured using cell lysates isolated from these cells. Wnt3a (100
ng/ml) was used as a positive control in this experiment. BIO significantly
increased the β-Gal activity (C). *P<0.05,
unpaired Student's t-test, n=3. Primary calvarial
osteoblasts were cultured with 0.1 and 1 μM of BIO for 2 days and changes
in ALP activity were examined by ALP staining. BIO (at both concentrations)
significantly increased ALP activity in these cells (D). To further determine
whether BIO induces new bone formation in vivo, 25 and 50 μg/mouse of BIO
was injected into 1-month-old WT mice subcutaneously over the surface of
calvariae for 5 days. Calcein labeling was performed at day 5 and 15. Mice
were sacrificed 2 days after the second calcein labeling and periosteal new
bone was evaluated and mineral appositional rates (MAR) were measured. FGF-1
(2 μg/mouse, 5 day injection) was used as a positive control. BIO
significantly increased new bone formation and MAR in this assay (E,F).
*P<0.05, one-way ANOVA followed by Dunnett's test (BIO
versus control) and unpaired Student's t-test (FGF-1 versus control),
n=5. All values are means ± s.e.