Effects of Angiopoietin-1 on Bone Morphogenetic Protein-2 Induced Osteoblast Differentiation

Objective: During bone formation and fracture healing, angiogenesis is observed before bone formation. However, roles of angiogenic factors in bone formation are not fully determined. This study was undertaken to examine whether angiopoietin-1 (Ang-1), an important modulator of vascular homeostasis and angiogenesis, is involved in bone morphogenetic protein (BMP)-2 induced osteoblast differentiation.

Methods: By RT-PCR analysis, expression of Ang-1 and its Tie-2 receptor mRNA was screened in osteoblast-like cell lines and primary mouse calvarial cell, and was also monitored during osteoblast differentiation induced by BMP-2 and/or ascorbic acid(AA) and b-glycerophosphate (GP). Roles of Ang-1 in osteoblast differentiation and mineralization were evaluated by ALP activity, Alizarin red stain, and OG2 and 6xOSE promoter assays. For molecular signaling mechanism, Western blot was done with MAPK and Akt antibodies.

Results: C3H10T1/2 and C2C12 cells showed strong expression of Tie-2 mRNA. Under the presence of BMP-2 and/or AA and b-GP, expression of Ang-1 and Tie-2 mRNA increased with induction of alkaline phosphatase (ALP) and osteocalcin (OC) mRNA in C3H10T1/2 and primary mouse calvarial cells. Overexpression of Ang-1 using adenoviral vectors significantly increased BMP2-induced ALP activity and OC production, as well as formation of mineralized nodules. Promoter study also revealed that Ang-1 stimulated the BMP-2 transactivation of OG2 and 6xOSE promoter. In Western blot analysis, Ang-1 enhanced BMP-2 induction of Akt phosphorylation in C3H10T1/2 cells.

Conclusion: These results suggest that Ang-1 enhance BMP-2 induced osteoblast differentiation, and that Akt pathway may be involved in BMP-2 signaling.

The study was supported by BK21 project for school of dentistry funded by the Korea government.