Molecular mechanisms of inhibitory effects of lactoferrin on LPS-mediated osteoclastogenesis

Objectives: Lactoferrin (LF) is an essential modulator of immune response and inflammation. In the previous study, we have demonstrated that oral administration of liposomal bovine LF (bLF) significantly reduced lipopolysaccharide (LPS)-induced alveolar bone resorption through reduction of tumor necrosis factor-α (TNF-α) production. However, it is not clear that how bLF inhibited LPS-mediated TNF-α production in osteoblasts and the direct effects of bLF on the osteoclasts differentiation. The aim of the study is to investigate the molecular mechanisms of inhibitory effects of bLF on LPS-mediated osteoclastogenesis. Methods: Gene and protein expressions were analyzed with real-time PCR system and Western blot analysis, respectively. Osteoclast formation was performed by single-culture and co-culture system. Results: bLF significantly inhibited LPS-mediated TNF-α and RANKL mRNA expressions in osteoblast-like cells, ST2. bLF inhibited nuclear factor kB (NFkB) activation by LPS. bLF also inhibited the mitogen-activated protein kinase (MAPK) activation. Remarkably, we found that bLF inhibited LPS-mediated TNF receptor-associated factor 6 (TRAF6) ubiquitination. Immunoprecipitates from bLF pretreated ST2 cells showed that bLF directly bound with endogenous TRAF6. Additionally, the intra cellular accumulation of bLF was inhibited by lipoprotein receptor related protein-1 (LRP1) knockdown. Moreover, bLF inhibited receptor activator of NFkB ligand (RANKL)-induced osteoclasts differentiation in preosteoclast linage cells, RAW. Notably, bLF treatment decreased the expression of RANK, which is the essential receptor for osteoclast differentiation. Conclusions: Present results suggest that endocytosed bLF via LRP1 exhibits the inhibitory effects on NFkB and MAPK pathways by interfering TRAF6 ubiquitination in osteoblasts, subsequently suppresses cytokine production and osteoclastogenesis via osteoblasts. Furthermore, bLF treatment directly decreases expression of RANK in preosteoclasts, resulting in the inhibition of osteoclasts differentiation. Collectively, bLF can be an effective inhibitory agent against bone destruction associated with inflammatory diseases like periodontitis.