Figure 3

Tumor necrosis factor (TNF) promotes the binding of nuclear proteins to the nuclear factor-kappa B (NF-κB) binding sequences of the vascular endothelial growth factor-C (VEGF-C) promoter. Raw264.7 osteoclast/macrophage precursors were cultured in 0.5% bovine serum albumin overnight. Cells were treated with TNF for 30 to 60 minutes. Nuclear extracts were isolated and subjected to Western blot analysis using anti-NF-κB p65 and p50 antibodies (a) or to electrophoretic mobility shift assay using a ³²P-labeled probe consisting of the putative NF-κB binding sequence of the mouse VEGF-C promoter (b). The specificity of binding was confirmed by using 50-fold more unlabeled wild-type (WT) or mutated probe in which the putative NF-κB binding sequence was mutated and could not be bound by NF-κB in a competition reaction. An SP-1 probe was used as a loading control. WT osteoclast precursors were treated with TNF ± an NF-κB inhibitor for 24 hours, and expression of VEGF-C was determined by real-time reverse transcription-polymerase chain reaction. Values are the means of three mice plus standard deviation. (c) The fold decrease in expression in the NF-κB inhibitor-treated over vehicle-treated cells was calculated. Experiments were repeated two times with similar results.