Inhibition of PI3-kinase activity blocks BMP2-induced osteoblast
differentiation. Results are shown of experiments in which C3H10T1/2 cells
were incubated in osteogenic media (OM) containing BMP2 for up to 10 days
without (Con, control) or with the MEK inhibitor UO126 (UO) (10 μM) or the
PI3-kinase inhibitor, LY294002 (LY) (20 μM), as described in the Materials
and Methods. (A) Immunoblots of whole-cell protein lysates for Akt
phosphorylated at Ser473 (pAktS473), total Akt, tyrosine and serine
phosphorylated Erk1 and Erk2 (pErk1/2), total Erks, serine phosphorylated
Smad1, Smad5 and Smad8 (pSmad1,5,8), total Smads and α-tubulin. (B)
Results of RT-PCR assays showing expression of osteoblast-specific genes
encoding Dlx-5, Runx2, osterix (Osx) and osteocalcin (Ocn), and the control
gene S17 after incubation for up to 7 days in osteogenic medium with or
without UO126 or LY294002. (C) Representative images of qualitative alkaline
phosphatase (AP) staining in cells after incubation in osteogenic medium with
or without UO126 or LY294002 for 5, 7 or 10 days. The graph depicts
measurement of alkaline phosphatase activity in lysates of cells incubated for
5 or 10 days in osteogenic medium with or without UO126 or LY294002 (mean
± s.d., n=3; *P<0.01,
**P<0.001 vs cells incubated without LY294002). (D)
Measurement of osteoblast-mediated mineralization assessed by Alizarin red
staining at days 5, 7 and 10 after incubation in osteogenic medium with or
without UO126 or LY294002. The graph shows calculation of mineralized area at
day 10 (mean ± s.d., n=5; **P<0.01 vs
cells incubated without LY294002).