The ultimate consequence of BMP-2 signaling is the activation of gene transcription, which promotes osteoblast differentiation and bone formation. Jeong et al. reported that GS-5734 COMP-Ang1 enhanced the BMP-2dependent transcriptional activity of the OG2 or 6xOSE promoters, indicating that the Ang1/Tie2 system might be involved in BMP-2-induced osteogenesis and modulation of BMP-2 target gene expression. They suggested that the effects of COMP-Ang1, MAPK, and PI3kinase/Akt signaling pathways may be a mechanism for the observed synergy. Tie2 phosphorylation followed by Ang1 binding to the receptor results in activation of PI3-kinase/Akt signaling pathway. The present study showed that COMP-Ang1 stimulated BMP-2-mediated induction of Akt phosphorylation as well as Smads and p38 MAPK phosphorylation, indicating that the synergy of COMP-Ang1 and BMP2 on osteoblast differentiation may be related to at least partial overlap of signaling pathways in osteoblasts. The positive effect of BMP-2 on osteoblast cells is well known; however, high failure rates and complications were reported when rhBMP-2 was used in treatment. In addition, TGF-b is responsible for the therapeutic resistance of BMP-2, as it causes BMP signaling interference. Transgenic mice lacking functional TGF-b signaling in osteoblasts or mice treated with the TGF-b type I receptor kinase inhibitor SD208 have increased trabecular bone mass with tougher femurs and stiffer and stronger vertebral bodies. These data suggest that continuous exposure to active TGF-b might harm bone physiology, as can be seen in patients suffering from chronic inflammation, whose active TGF-b1 serum levels are often constantly elevated. In this study, serum TGF-b1 levels were increased 4-fold two weeks after surgery in all four animal groups. One possible mechanism by which TGF-b1 may exert its inhibitory effect on osteoblast differentiation is through interference with BMP signaling. In this study, COMP-Ang1 decreased phosphorylation of Smad2/3, a downstream protein of TGF-b1 pathways. Therefore, these results suggest that COMP-Ang1 inhibits the TGF-b1 signaling pathway in addition to enhancing the BMP-2 signaling pathway. According to previous studies, the Tie2 receptor, a target of COMP-Ang1, is expressed in quiescent hematopoietic stem cells of the bone marrow. Our study showed that the Tie2 receptor is expressed in C3H10T1/2 cells. Based on these findings, we hypothesized that COMP-Ang1 also plays a direct role in osteoblast differentiation through the Ang1-Tie2 pathway. Meanwhile, COMP-Ang1 interacts with integrins and promotes endothelial cell survival. Integrins work as extracellular matrix receptors that transduce signals from the environment into the cell interior.