Role of vasodilator-stimulated phosphoprotein in RANKL-differentiated murine macrophage RAW264.7 cells: Modulation of NF-κB, c-Fos and NFATc1 transcription factors.

Vasodilator-stimulated phosphoprotein (VASP) is essential for osteoclast differentiation, and reduced VASP expression results in depressed osteoclast differentiation. Previously, we demonstrated the importance of VASP and Ras-related C3 botulinum toxin substrate 1 interactions in osteosarcoma cell migration and metastasis using Mg-63 and Saos2 cells. However, the molecular details of the functional role of VASP in cell motility and migration remain to be elucidated. The present study demonstrated that VASP affects the expression of αV-integrin, tartrate-resistant acid phosphatase (TRAP) and lamellipodia protrusion in RAW 264.7 murine macrophage cells. The RAW 264.7 mouse monocyte macrophage cell line was used as an osteoclast precursor. RAW 264.7 cells were treated with 50 ng/ml of receptor activator of nuclear factor κ-Β ligand (RANKL) in order to induce cell differentiation (osteoclastogenesis). Small interfering RNA (siRNA) was used to silence VASP, and RT-PCR and western blotting were used to determine the expression for genes and proteins, respectively. TRAP staining as a histochemical marker for osteoclast and fluorescent microscopy for lamellipodia protrusion was performed. RANKL treatment significantly increased the gene and protein expression of VASP, αV-integrin and TRAP in RAW 264.7 cells. Silencing of VASP significantly reduced the RANKL-induced expression of αV-integrin, TRAP and lamellipodia protrusion. In addition, knockdown of VASP attenuated RANKL-stimulated activation of NF-κB, c-Fos and nuclear factor of activated T cells cytoplasmic 1 transcription factors, and the phosphorylation of the p65 and IκBα. These results suggest the critical role of VASP in regulating osteoclast differentiation, which should be further explored in osteosarcoma research.