Abstract
Osterix (Osx) is an osteoblast-specific transcription factor required for bone formation and osteoblast differentiation. The critical step in bone formation is to replace the avascular cartilage template with vascularized bone. Osteogenesis and angiogenesis are associated with each other, sharing some essential regulators. Vascular endothelial growth factor (VEGF) is involved in both angiogenesis and osteogenesis. Transcriptional regulation of VEGF expression is not well known in osteoblasts. In this study, quantitative real-time RT-PCR results revealed that VEGF expression was down-regulated in Osx-null calvarial cells and that osteoblast marker osteocalcin expression was absent. Overexpression of Osx in stable C2C12 mesenchymal cells using a Tet-off system resulted in up-regulation of both osteocalcin and VEGF expression. The inhibition of Osx by siRNA led to repression of VEGF expression in osteoblasts. These results suggest that Osx controls VEGF expression. Transfection assays demonstrated that Osx activated VEGF promoter activity. A series of VEGF promoter deletion mutants were examined and the minimal Osx-responsive region was defined to the proximal 140-bp region of the VEGF promoter. Additional point mutants were used to identify two GC-rich regions that were responsible for VEGF promoter activation by Osx. Gel shift assay showed that Osx bound to the VEGF promoter sequence directly. Chromatin immunoprecipitation assays indicated that endogenous Osx associated with the native VEGF promoter in primary osteoblasts. Moreover, immunohistochemistry staining showed decreased VEGF protein levels in the tibiae of Osx conditional knock-out mice. We provide the first evidence that Osx controlled VEGF expression, suggesting a potential role of Osx in coordinating osteogenesis and angiogenesis.
Highlights
Osx is an osteoblast-specific transcription factor required for bone formation and osteoblast differentiation
Vascular endothelial growth factor (VEGF) mRNA Levels Are Down-regulated in the Absence of Osx—Angiogenesis plays an important role in bone formation
Osx is an osteoblast-specific transcription factor required for osteoblast differentiation and bone formation
Summary
Osx is an osteoblast-specific transcription factor required for bone formation and osteoblast differentiation. Results: Osx directly targets vascular endothelial growth factor expression, whereas Osx controls osteoblast marker gene osteocalcin. Significance: This is the first evidence that Osx controls vascular endothelial growth factor expression, suggesting a potential role of Osx in coordinating osteogenesis and angiogenesis. Osterix (Osx) is an osteoblast-specific transcription factor required for bone formation and osteoblast differentiation. Endochondral ossification is responsible for the formation of most bones, such as long bones, and requires a cartilage intermediate Fewer bones, such as craniofacial bones are formed by intramembranous ossification, in which bones form directly from mesenchymal condensations. Osteoblast differentiation from mesenchymal stem cells is controlled by various transcription factors and signaling proteins, including Indian hedgehog, Runx, Osterix (Osx), and the Wnt/-catenin signaling pathway [1]. Our recent observation that Osx inhibits the Wnt signaling pathway highlights the
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