VEGF165 promotes the osteolytic bone destruction of ewing's sarcoma tumors by upregulating RANKL

Abstract

The purpose of this study was to determine whether vascular endothelial growth factor-165 (VEGF165) contributed to the osteolytic process in Ewing's sarcoma. VEGF165 induced osteoclast formation from murine bone marrow cells. Tartrate-resistant acid phosphatase (TRAP) staining demonstrated significantly fewer osteoclasts in VEGF-inhibited TC/siVEGF7-1 tumors compared to TC71 parental or TC/si-control tumors. Receptor activator NF-kappaB (RANKL), a critical osteoclastogenic factor, was decreased in TC/siVEGF7-1 cells. Incubation of these cells with recombinant VEGF165 upregulated RANKL in a dose- and time-dependent manner. The induction of (RANKL) by VEGF165 was also demonstrated in MC3T3-E1 mouse osteoblast cells and bone marrow stromal cells. This upregulation was transcriptionally mediated by an effect on the RANKL promoter. Both VEGF and EWS/FLI-1 increased RANKL promoter activity. Taken together, these data suggest that modulation of RANKL by VEGF165 may be one of the mechanisms responsible for the osteolytic process induced by Ewing's sarcoma cells. VEGF165 may, therefore, play an important role in modulating RANKL gene expression in the bone marrow microenvironment during the metastatic process, thereby contribution to tumor induced bone lysis.

Figure 1

Effect of VEGF on TC71 tumor phenotype and osteoclast formation. (A) TC71, TC/si-control, or TC/siVEGF_7-1 cells were injected into the tibia of nude mice. Bone tumors were removed after 3 weeks and assessed by H&E staining. The presence of osteoclasts was determined by TRAP staining (arrows). (B) Murine bone marrow cells were cultured with rhVEGF or rhM-CSF for 9 days. Osteoclast formation was assessed by TRAP staining. (C) TRAP-positive multinuclear cells were quantified by light microscopy. (D) Suspended murine bone marrow cells were cultured with rhVEGF or rhM-CSF in the presence or absence of murine osteoblast MC3T3-E1 cells. TRAP-positive multinuclear cells were then quantified. Results are the mean ± SE of 3 separate experiments (P<0.05).

Figure 2

Effect of VEGF on RANKL expression in murine bone marrow stromal cells and osteoblasts. Serum-starved murine bone marrow stromal cells (A) or MC3T3-E1 murine osteoblast cells (B) were cultured with rhVEGF 100 ng/ml for 2 h. Total RNA was extracted, and RANKL expression was quantified by RT-PCR. (C) MC3T3-E1 murine osteoblast cells were cultured with rhVEGF at different concentrations for 24 h. RANKL protein level was quantified by Western blot analysis. (D) MC3T3-E1 cells were transfected with 1 μg of RANKL/Luc reporter plasmid plus 50 ng of Renilla-luc expression vector. Twenty-four h after transfection, cells were serum starved overnight and then stimulated with rhVEGF 100 ng/ml for 3, 7, and 24 h. Luciferase activity was determined. Results are expressed as relative luciferase activity normalized to Renilla luciferase activity from the transfection control plasmid and represent the mean value ± SE from 3 experiments (P<0.05).

Figure 3

Effect of VEGF on RANKL expression in TC71 parental and transfected cells. (A) Cell lysates were extracted from TC71, TC/si-, TC/siVEGF_7-1 and TC/siVEGF_7-17 cells and assayed by Western blot analysis using anti-human RANKL antibody. β-actin was used as the internal control. (B, C) Serum-starved TC/siVEGF_7-1 cells were cultured with (B) various concentrations of rhVEGF for 24 h, or (C) with rhVEGF at 100 ng/ml for different time points. Cell lysates were collected and assayed by Western blot analysis.

Figure 4

VEGF upregulates RANK. (A) Serum starved RAW246.7 cells were cultured with rhVEGF at 100ng/ml for 2 h. RANK expression was determined by RT-PCR.

Figure 5

EWS/FLI-1 regulates RANKL expression in TC71 Ewing's sarcoma cells. (A) Murine fibroblast NIH3T3 cells were stably transfected with EWS/FLI-1 expression plasmid. RANKL protein level was determined by Western blot analysis. β-actin was the internal control. (B) TC71 cells were transiently transfected with EWS/FLI-1 siRNA. RANKL protein levels were determined by Western blot analysis following transfection. (C) NIH 3T3 cells were transfected with pGL3 luciferase vector (control), or RANKL promoter/luc vector (RANKL promoter 2 kb or 4 kb), with or without cotransfection of EWS/FLI-1-expression plasmid. Cell lysates were collected 24h later and luciferase activity quantified. (D) Serial deletion RANKL promoter plasmids were constructed. NIH 3T3 cells were co-transfected with EWS/FLI-1 expressing plasmid and pGL3 luciferase reporting plasmid, RANKL-luciferase plasmid or one of serial deletion constructs. Cell lysates were collected 24 h later and luciferase activity quantified.