J Korean Orthop Assoc.  2007 Aug;42(4):537-544. 10.4055/jkoa.2007.42.4.537.

Synergistic Effect of Isoflavones on the BMP-4 MediatedOsteoblastic Differentiation of C2C12 Myoblast

• Cui HS
• Kim YM
• Lee SJ
• Choi JK
• Kim DS
• Choi ES
• Shon HC
• Park KJ
• Lee HK

• KMID: 2186517
• DOI: http://doi.org/10.4055/jkoa.2007.42.4.537

Abstract

PURPOSE: Isoflavones are rich in soybean and are known to affect bone formation. This study examined the effects and modes of action of isoflavones on the differentiation of C2C12 myoblasts in the presence of the bone morphogenetic protein (BMP)-4.
MATERIALS AND METHODS
The isoflavones, daidzein, genistein or equol, and/or BMP-4 were added alone or in combination to C2C12 myoblasts. After 72 hours culture, the cells were stained for the early osteoblastic differentiation marker, alkaline phosphatase (ALP). The ALP activity was determined by comparing the color of the stained images as well as by spectrophotometry. The expression profiles of the extracellular matrix (ECM) genes responsible for the extensive remodeling at the cell surface were analyzed using agene expression microarray after treating thesamples with daidzein.
RESULTS
ALP staining of BMP-4 or the isoflavones-treated cells showed that BMP-4 increased the activity of ALP in a dose dependent manner, whereas the isoflavones alone did not induced any remarkable increase. However, the ALP activity increased when the cells were treated with BMP-4 and any of the three isoflavones. The macrogen mouse MAC array data showed that the ECM genes, Mmp13 and Mmp3, were up-regulated by daidzein, whereas Col4a2, Col5a1 and Mmp9 were down-regulated.
CONCLUSION
Isoflavones induce osteoblastic differentiation when combined with BMP-4, which is possibly achieved by modulating the expressional levels of various ECM genes.

Keyword

Isoflavone; Osteoblastic differentiation; C2C12; BMP-4

MeSH Terms

Alkaline Phosphatase
Animals
Bone Morphogenetic Proteins
Equol
Extracellular Matrix
Genistein
Isoflavones*
Mice
Myoblasts*
Osteoblasts
Osteogenesis
Soybeans
Spectrophotometry
Alkaline Phosphatase
Bone Morphogenetic Proteins
Equol
Genistein
Isoflavones

Figure

• 

  Fig. 1 ALP Activity of C2C12 cellswas induced effectively by daidzein and BMP-4.

• 

  Fig. 2 (A-C) ALP activities of C2C12 cells were strongly induced by isoflavones (esp. daidzein) in the presence of various BMP-4 concentrations (results by spectrophotometry).

• 

  Fig. 3 Gene expressions of the ECM structural proteins were down-regulated in response to daidzein (1=daidzein; 2=control).

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