acid-phosphatase and phenylalanyl-leucyl-phenylalanyl-glutaminyl-prolyl-glutaminyl-arginyl-phenylalaninamide

Neuropeptide FF (NPFF) has been implicated in many physiological processes but not osteoclastogenesis. We previously demonstrated that NPFF modulates the viability and nitric oxide (NO) production of RAW264.7 macrophages. This study was designed to investigate the effect of NPFF on receptor activator of nuclear factor κB ligand (RANKL)-mediated differentiation of RAW264.7 cells into osteoclast-like cells.. RAW264.7 cells were cultured in 96-stripwell plates or in Corning Osteo Assay Surface 96-well plates in the presence of various concentrations of NPFF with or without RANKL for 3 or 6 d. The differentiation of osteoclast-like cells was analyzed by tartrate-resistant acid phosphatase (TRAP) stain, TRAP activity and bone resorption capacity, respectively. The mRNA expression of NPFF2 receptor (NPFFR2) and osteoclast genes was evaluated by using real-time quantitative PCR which includes TRAP, RANK (receptor activator of NF-κB), Cathepsin K, MMP-9 (matrix metallopeptidase 9), Intβ3 (integrin β3) and NFATc1 (nuclear factor of activated T cells cytoplasmic 1). In addition, the influence of NPFF on the cell viability and NO release of RAW264.7 cells was measured by MTT assay and Griess method, respectively.. NPFF dose-dependently inhibited RANKL-induced osteoclast-like cells differentiation including TRAP-positive cell formation, TRAP activity and bone resorption capacity. Moreover, NO release and osteoclast gene expression of osteoclast-like cells were downregulated by NPFF. In addition, NPFFR2 gene expression in osteoclast-like cells was augmented in response to NPFF treatment.. Our findings showed that NPFF could attenuate osteoclast-like cells differentiation in an in vitro osteoclastogenesis model.

Topics: Acid Phosphatase; Bone Resorption; Cell Differentiation; Cell Line; Cells, Cultured; Down-Regulation; Isoenzymes; Macrophages; Oligopeptides; Osteoclasts; RANK Ligand; Real-Time Polymerase Chain Reaction; Tartrate-Resistant Acid Phosphatase

Neuropeptide FF (NPFF) has been implicated in many physiological processes. Previously, we have reported that NPFF modulates the viability and nitric oxide (NO) production of RAW264.7 macrophages. In this study, we investigated the influence of NPFF on lipopolysaccharide (LPS)-mediated osteoclast formation of RAW264.7 cells. Our results suggest that, NPFF dose-dependently (1 nM, 10 nM and 100 nM) inhibited osteoclast formation, TRAP enzyme activity and bone resorption in osteoclasts induced by LPS respectively. Moreover, LPS-provoked NO release was also inhibited by NPFF treatment, indicating a NO-dependent pathway is mainly involved. Furthermore, the alterations of osteoclast marker genes were also assessed including TRAP, Cathepsin K, MMP-9, NFATc1 and Runx2. NPFF downregulated LPS-caused gene augmentations of TRAP, Cathepsin K and MMP-9, whereas showed no influences on NFATc1 and Runx2. In addition, NPFF receptor 2 (NPFFR2) mRNA expression was also augmented in response to NPFF treatment, hinting the involvement of NPFFR2 pathway. It should be mentioned that RF9 (1 µ M), a reported pharmacological inhibitor for NPFF receptors, exerted NPFF-like agonist properties as to attenuate osteoclastogenesis. Collectively, our findings provide new evidence for the in vitro activity of NPFF on osteoclasts, which may be helpful to extend the scope of NPFF functions.

Topics: Acid Phosphatase; Adamantane; Animals; Cell Differentiation; Cell Line; Dipeptides; Gene Expression Regulation; In Vitro Techniques; Isoenzymes; Lipopolysaccharides; Macrophages; Mice; Oligopeptides; Osteoclasts; Receptors, Neuropeptide; Tartrate-Resistant Acid Phosphatase; Up-Regulation