Introduction: The aim of this study is to determine whether regulation of the expression level of fms-like tyrosine kinase-4 (Flt-4) is related to osteoclast differentiation.
Material and methods: Osteoclast formation and differentiation of mouse bone marrow cells and RAW264.7 cells were performed. To induce osteoclast differentiation, RANKL (50 ng/ml) with or without vascular endothelial growth factor-C (VEGF-C) and vascular endothelial growth factor-D (VEGF-D) was added to mouse bone marrow cells and RAW264.7 cells. Then cells were examined under a microscope. TRAP-positive cells with 3 nuclei or more were considered as osteoclasts and counted. The Flt-4 gene was knocked down by transfection of siRNAs against Flt-4. Immunoblot analyses were performed.
Results: The osteoclast formation assay indicated that VEGF-C resulted in 500 or 450 vs. 100 (p < 0.05) of osteoclasts in mouse bone marrow cells and RAW264.7 cells, respectively. Vascular endothelial growth factor-D resulted in about 600 or 630 vs. 100 (p < 0.05) of osteoclasts for both mouse bone marrow cells and RAW264.7 cells. The knock-down of Flt-4 expression abolished the induction by VEGF-C or VEGF-D, resulting in induction similar to that of the negative control PBS.
Conclusions: Both VEGF-C and VEGF-D can induce osteoclast differentiation in the presence of the receptor activator of nuclear factor -B ligand. Down-regulation of expression level of Flt-4 protein abolishes osteoclast differentiation induced by VEGF-C or VEGF-D.