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Neuropeptide FF inhibits LPS-mediated osteoclast differentiation of RAW264.7 cells
Abstract:
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.
Insights
Neuropeptide FF (NPFF) inhibits osteoclast formation and bone resorption in a dose-dependent manner, potentially through a nitric oxide (NO)-dependent pathway involving NPFF receptor 2 (NPFFR2). This suggests novel roles for NPFF in bone metabolism.
Area of Science:
- Immunology
- Endocrinology
- Cell Biology
Background:
- Neuropeptide FF (NPFF) is known to influence various physiological functions.
- Previous research indicated NPFF modulates RAW264.7 macrophage viability and nitric oxide (NO) production.
Purpose of the Study:
- To investigate the effect of NPFF on lipopolysaccharide (LPS)-induced osteoclast formation in RAW264.7 cells.
- To explore the underlying mechanisms, including NO involvement and gene expression changes.
Main Methods:
- RAW264.7 macrophages were treated with NPFF and LPS to assess osteoclastogenesis.
- Measurements included osteoclast formation, TRAP enzyme activity, bone resorption, NO release, and expression of osteoclast marker genes (TRAP, Cathepsin K, MMP-9, NFATc1, Runx2).
- NPFF receptor 2 (NPFFR2) mRNA expression and the effect of RF9 (NPFF receptor antagonist) were also evaluated.
Main Results:
- NPFF dose-dependently inhibited LPS-induced osteoclast formation, TRAP activity, and bone resorption.
- NPFF suppressed LPS-stimulated NO release, suggesting a NO-dependent mechanism.
- NPFF downregulated LPS-induced expression of TRAP, Cathepsin K, and MMP-9, but not NFATc1 or Runx2.
- NPFF treatment increased NPFFR2 mRNA expression, and RF9 exhibited NPFF-like inhibitory effects on osteoclastogenesis.
Conclusions:
- NPFF exhibits inhibitory effects on osteoclast formation and function in vitro.
- The mechanism likely involves a NO-dependent pathway and modulation of specific osteoclast marker genes via NPFFR2.
- These findings expand the known physiological roles of NPFF, particularly in bone metabolism.
