Transcriptional regulation of Vascular Endothelial Growth Factor (VEGF) by osteoblast-specific transcription factor Osterix (Osx) in osteoblasts

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.

FIGURE 1.

Effect of Osx on VEGF expression. A, in the absence of Osx, osteocalcin (OC), and VEGF mRNA levels were down-regulated. Fold-change in RNA levels is shown. Calvaria RNAs were from E18.5 Osx wild-type and Osx-null embryos. RNA expression levels for Osx, osteocalcin, Runx2, and VEGF were analyzed by real-time RT-PCR. The level of each RNA from Osx-null calvaria was normalized to a value of 1. Values are presented as the mean ± S.D. B, overexpression of Osx activates VEGF gene expression in C2C12 mesenchymal cells. Osx expression is induced in the absence of Dox in this cell line. RNA was obtained from cultures treated with or without Dox. VEGF and OC mRNA levels were quantitated by real-time RT-PCR. The VEGF RNA level obtained from the cells cultured with Dox was normalized to a value of 1. Fold-change in RNA levels is shown. Values are presented as the mean ± S.D.

FIGURE 2.

siRNA-directed knockdown of Osx impairs VEGF gene expression in MC3T3 osteoblasts. A, RNA expression levels were determined by quantitative real-time RT-PCR. MC3T3 osteoblasts were transfected with siRNA targeting mouse Osx. RNA was isolated 24 h post-transfection and quantitated by real-time RT-PCR. The RNA level from the control siRNA group was normalized to a value of 1. Values were presented as the mean ± S.D. B, Western analysis of the Osx knockdown. Protein was isolated by acetone precipitation of whole cell lysates and then analyzed by Western blot using rabbit anti-VEGF or anti-Osx polyclonal antibodies. β-Actin was used as a loading control.

FIGURE 3.

Identification of the Osx binding site in the promoter of VEGF gene. A, Osx regulates VEGF promoter activities in a dose-dependent manner. HEK293 cells were plated in 12-well tissue culture dishes. Each group of the cells was transiently transfected with 300 ng of promoter-luciferase reporter gene and the indicated amount of osterix expression vector. Cells were analyzed 24 h after transfection. Luciferase activity was normalized by β-galactosidase activity. Values are presented as the mean ± S.D. B, deletion analysis of the VEGF promoter-reporter constructs. VEGF-1kb, VEGF-500bp, VEGF-270bp, and VEGF-140bp promoter-reporter plasmids (300 ng each) were cotransfected with 400 ng of the Osx expression plasmid in HEK293 cells. After 24 h transfection, cell extracts were prepared and analyzed for luciferase activity and normalized to β-galactosidase activity. C, two GC-rich elements in VEGF-140bp are responsible for VEGF promoter reporter activation by Osx. VEGF-M1, VEGF-M2, and VEGF-M12 are individual mutants of the VEGF-140bp construct. They were transfected as described in panel B. Luciferase activity was normalized by β-galactosidase activity. D, a diagram of the proximal 140-bp region of the mouse VEGF promoter. M1 refers to point mutations of VEGF-M1, and M2 refers to point mutations of VEGF-M2. VEGF-M12 in C contains both M1 and M2.

FIGURE 4.

Osx associates with VEGF promoter. A, Osx bound to VEGF promoter oligos in a gel shift assay. DNA oligonucleotides of VEGF wild-type (WT) and mutant were labeled by Biotin. Osx protein and the biotin-labeled DNA probe were incubated. Protein-DNA complexes were separated on 5% polyacrylamide gels, and visualized by a chemiluminescent nucleic acid detection module. VEGF WT oligos were incubated with control buffer (lane 1) or Osx (lane 2). VEGF mutant oligos (lane 3) were used. Baculovirus-expressed Osx was used as the protein resource. B, endogenous Osx associated with native VEGF promoter in vivo analyzed by quantitative real-time PCR. The ChIP assay was carried out in primary calvarial osteoblasts isolated from newborn wild-type mice. Anti-Osx antibody was used for ChIP analysis, and IgG was used as a negative control. The precipitated chromatin was analyzed by quantitative real-time PCR. Primer set 1 corresponds to a segment covering two GC-rich elements within 140 bp the VEGF promoter. As a negative control, primer set 2 covers a distal 1-kb region of the VEGF promoter, which does not contain GC-rich sequence.

FIGURE 5.

VEGF protein level is decreased in osteoblasts of conditional Osx-null mice. A, immunohistochemistry analysis using anti-VEGF antibody in tibiae of tamoxifen-treated wild-type mice. B, immunohistochemistry analysis using anti-IgG antibody in tibiae of tamoxifen-treated wild-type mice. C, immunohistochemistry analysis using anti-VEGF antibody in tibiae of tamoxifen-treated Osx^postnatal mutant mice. Immunohistochemistry was performed with VEGF antibody or IgG on the decalcified paraffin sections of tibiae of tamoxifen-treated 6-week-old mice. Representative images of immunohistochemistry sections of tibiae were shown. Wt/Tam, tamoxifen-treated wild-type mice; Null/Tam, tamoxifen-treated Osx^postnatal mutant mice.