Fig 1.
Expression of Rap1A during osteoblastic differentiation.
(a) Endogenous expression of Rap-1A in MC3T3-E1 and C2C12 cells by Western blotting. (b) The temporal change of Rap1A expression in MC3T3-E1 cells during osteoblastic differentiation by Western blotting. (c) Alizarin red staining for bone nodules in MC3T3-E1 cells. Original magnification was ×20 (left). Arizalin red-S staining activity was quantified by densitometry at 562 nm (right). Data represent means ± SD of triplicate samples. *P < 0.05, **P < 0.01 vs. the undifferentiated cells. β-actin was used as the internal control.
Fig 2.
Association of Rap1A with osteoblast differentiation.
(a) Immunoblot analysis of Rap1A expression during osteogenic differentiation. The protein extracts were immunoblotted with anti-Rap1A antibody. β-Actin was used as the internal control. (b-f) Changes of osteoblastic markers gene expression in differentiated C2C12 cells. C2C12 cells were cultured with osteogenic medium containing ascorbic acid (50μg/ml), and β-glycerophosphate (10 mM) for the indicated times. Quantitative real-time PCR was performed for the mRNA expression of Rap1A, Runx2, Col1a1, Osteocalcin, and Osterix. (g) Alkaline phosphatase (ALP) staining and ALP activity measurement were determined during osteoblastic differentiation from C2C12 cells. (h) Osteoblastic mineralization of C2C12 cells was determined by Alizarin red staining and calcium content quantified in the cellular matrix by spectrophotometer (OD562nm). Data represent means ± SD of triplicate samples. *P<0.05, **P<0.01 vs. the undifferentiated cells.
Fig 3.
RalGDS pull-downs to assess Rap1A activity induced by osteogenic stimulation.
C2C12 and MC3T3-E1 cells were treated with osteogenic inducers (AA/β-GP) for the indicated times and subjected to GST-RalGDS pulldown assays to detect the active form of Rap1A. The levels of captured GTP-Rap1A were determined by immunoblot analysis using an anti-Rap1A antibody. Levels of total Rap1A and β-Actin (loading control) in whole-cell lysates were also determined by immunoblot analysis.
Fig 4.
Knockdown of Rap1A inhibits osteoblast differentiation and osteogenic gene expression.
(a) Representative clones expressing shRNA against Rap-1A were analyzed for Rap1A expression by immunoblot. β-actin was used as a loading control. (b) Proliferation of C2C12 cells with or without shRap1A. Relative absorbance of cells in MTT assay was determined at daily intervals over 4 days' incubation. (c) Knockdown of Rap1A in C2C12 cells reduced ALP activity. Cells cultured for 10 d with or without osteogenic inducers (AA/β-GP) were fixed and stained for ALP. ALP activities were measured by densitometry at 520 nm (below). (d) Knockdown of Rap1A in C2C12 cells reduced mineralized matrix formation. Cells cultured for 14 d with or without osteogenic inducers (AA/β-GP) were fixed and stained for Alizarin red and photographed. The eluted alizarin red stain was quantified by densitometry at 562 nm (below). (e-i) Knockdown of Rap1A expression in C2C12 cells suppressed osteoblast-specific gene expression. Transduced cells were cultured with osteogenic medium containing ascorbic acid (50 μg/ml), and β-glycerophosphate (10 mM) for the indicated times and quantitative real-time PCR analysis of mRNA expression of Rap1A, Runx2, Col1a1, Osteocalcin, and Osterix was performed. (b-i) Data represent means ± SD of triplicate samples. *, P < 0.05; **, P < 0.01 versus control.
Fig 5.
Silencing of Rap1A decreases ERK and p38 activation.
Representative immunoblots for Rap1A expression and MAPK activation in control and Rap1A shRNA expressing C2C12 cells grown in basal or osteogenic medium containing ascorbic acid (50 μg/ml), and β-glycerophosphate (10 mM) for 6 and 10 d. Western blot analyses with antibodies recognizing Rap1A, phospho-ERK1/2, total ERK1/2, phospho-p38 and total p38 were performed. β-actin was used for internal control.
Fig 6.
Forced expression of Rap1A accelerates osteoblast differentiation through activation of ERK1/2 and p38 MAPK.
(a) 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) Proliferation of MC3T3-E1 cells with or without Rap1A overexpression tested by MTT assays. (c-g) Over-expression of Rap1A in MC3T3-E1 cells increased osteoblast-specific gene expression. (h) 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. (i) 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. (j and k) 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 (j) or 10 μM SB203580 (k). 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.