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The Role for SDF-1/CXCR4 Signal Axis in BMP9-induced Osteogenic Differentiation of C2C12 Cells
Liu Chen, Yang Dandan, Bai Huili, Li Baolin, He Fang, Zhang Ruyi, Yan Shujuan, Shi Qiong*
Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Faculty of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China
Abstract: To elucidate the role of SDF-1/CXCR4 signal axis during BMP9-incuced osteogenic differentiation
in C2C12 cells， BMP9 was introduced by using recombinant adenoviruses assay. The mRNA and protein
expression levels of SDF-1 and CXCR4 induced by Ad-BMP9 were detected in C2C12 cells. At the same time， the
recombinant adenovirus and neutralizing antibody were used to perturbing the SDF-1/CXCR4 signal axis in C2C12
cells before or after the addition of BMP9. The alkaline phosphatase (ALP) quantitative assay and fast blue RR salt
staining were used to determine the expression of ALP. Immunocytochemistry was used to determine the expression
of osteocalcin (OCN)， while calcium deposition was determined by alizarin red S staining. The expression of
the osteogenic transcription factor Runx2 and Osx were detected by real time PCR. Western blot was used to detect
the change of osteogenic differentiation signaling pathway MAPK and Smad. The results showed that BMP9 significantly inhibited SDF-1 and CXCR4 expression (P<0.01) in C2C12 cells， in a dose- and time-dependent.
Pretreatment of C2C12 cells with SDF-1/CXCR4 could significantly affect the early and mid osteogenic markers
ALP， OCN， the transcription factors of Runx2， Osx expression (P<0.01)， and the Smad， MAPK signaling pathway
(P<0.05). Addition of exogenous SDF-1 did not affect the changes of the late osteogenic marker calcium deposition.
Our data indicated a co-requirement of the SDF-1/CXCR4 signal axis in BMP9-induced the early- and midprocess
of osteogenic differentiation of C2C12 cells.
in C2C12 cells， BMP9 was introduced by using recombinant adenoviruses assay. The mRNA and protein
expression levels of SDF-1 and CXCR4 induced by Ad-BMP9 were detected in C2C12 cells. At the same time， the
recombinant adenovirus and neutralizing antibody were used to perturbing the SDF-1/CXCR4 signal axis in C2C12
cells before or after the addition of BMP9. The alkaline phosphatase (ALP) quantitative assay and fast blue RR salt
staining were used to determine the expression of ALP. Immunocytochemistry was used to determine the expression
of osteocalcin (OCN)， while calcium deposition was determined by alizarin red S staining. The expression of
the osteogenic transcription factor Runx2 and Osx were detected by real time PCR. Western blot was used to detect
the change of osteogenic differentiation signaling pathway MAPK and Smad. The results showed that BMP9 significantly inhibited SDF-1 and CXCR4 expression (P<0.01) in C2C12 cells， in a dose- and time-dependent.
Pretreatment of C2C12 cells with SDF-1/CXCR4 could significantly affect the early and mid osteogenic markers
ALP， OCN， the transcription factors of Runx2， Osx expression (P<0.01)， and the Smad， MAPK signaling pathway
(P<0.05). Addition of exogenous SDF-1 did not affect the changes of the late osteogenic marker calcium deposition.
Our data indicated a co-requirement of the SDF-1/CXCR4 signal axis in BMP9-induced the early- and midprocess
of osteogenic differentiation of C2C12 cells.
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