Forced expression of Rap1A accelerates osteoblast differentiation through activation of ERK1/2 and p38 MAPK.

<p><b>(a)</b> MC3T3-E1 cells stably expressing Rap1A were established as stated in the ‘‘Materials and Methods,” and Western blotting was used to test Rap1A expression with antibodies recognizing HA and Rap1A. <b>(b)</b> Proliferation of MC3T3-E1 cells with or without Rap1A overexpression tested by MTT assays. <b>(c-g)</b> Over-expression of Rap1A in MC3T3-E1 cells increased osteoblast-specific gene expression. <b>(h)</b> Western blot analyses with antibodies recognizing phospho-ERK1/2, total ERK1/2, phospho-p38, total p38 and Rap1A were performed in cells treated with either growth medium (GM) or osteoblast differentiation medium (DM) containing rhBMP-2 (100 ng/ml), ascorbic acid (50 μg/ml), and β-glycerophosphate (10 mM) for 3 days. β-actin was used as a loading control. <b>(i)</b> phospho-ERK1/2, total ERK1/2 and Rap1A were detected by Western blot in MC3T3-E1 cells with or without Rap1A overexpression after treatment with osteoblast differentiation medium (DM) for 3 days in the presence or absence of 10 μM U0126. β-actin was used as a loading control. <b>(j and k)</b> Inhibition of ALP activity by U0126 and SB203580 in cells with or without Rap1A overexpression after differentiation induction by osteogenic medium containing rhBMP-2 (100 ng/ml), ascorbic acid (50 μg/ml), and β-glycerophosphate (10 mM) for 3 days in the presence or absence of 10 μM U0126 <b>(j)</b> or 10 μM SB203580 <b>(k)</b>. Cells were fixed and stained for ALP. ALP activities were measured by densitometry at 520 nm (below). Data represent means ± SD of triplicate samples. *, P < 0.05; **, P < 0.01.