Bone formation is a highly regulated process involving the differentiation of mesenchymal stem cells to osteoblasts. Angiogenesis and osteogenesis are tightly coupled during bone formation. Vascular endothelial growth factor (VEGF) is involved in both processes. Relatively little is known about VEGF gene regulation in osteoblasts. Osterix (Osx) is a bone morphogenetic protein 2 (BMP-2) inducible osteoblast-specific transcription factor required for osteoblast differentiation and bone formation. Our recent study has demonstrated that Osx controls VEGF expression in osteoblasts. Here, we further characterized Osx regulation of VEGF. To address which domain of Osx is responsible for VEGF regulation, the deletion mutant analysis and transfection assay were carried out to show that proline-rich region (PAR) is required for Osx activation of VEGF promoter activity. Hypoxia-inducible factor-1 alpha (HIF-1 alpha) has been reported to couple angiogenesis to osteogenesis, and to upregulate VEGF. Effect of Osx on HIF-1 alpha expression was examined in this study. Quantitative RT-PCR results revealed that HIF-1 alpha remained unchanged between wild type and Osx knockout calvaria at E18.5 in mouse embryos. Overexpression of Osx in stable C2C12 mesenchymal cells using Tet-off system did not affect HIF-1 alpha expression. HIF-1 alpha level did not change after Osx inhibition by siRNA in osteoblasts. Moreover, BMP-2 stimulation led to upregulation of Osx and VEGF, but not HIF-1 alpha. These results demonstrate that HIF-1 alpha is not a downstream target of Osx in osteoblasts, suggesting that Osx regulation of VEGF is independent of HIF-1 alpha expression level. Interestingly, synergistic interplays were observed between Osx and HIF-1 alpha in VEGF promoter activation in transfection assay. Our findings indicate that Osx and HIF-1 alpha cooperatively regulate VEGF expression. (C) 2012 Elsevier Inc. All rights reserved.