Molecular Mechanisms of the Inhibitory Effects of Bovine Lactoferrin on Lipopolysaccharide-mediated Osteoclastogenesis

Lactoferrin (LF) is an important modulator of the immune response and inflammation. It has also been implicated in the regulation of bone tissue. In our previous study we demonstrated that bovine LF (bLF) reduces LPS-induced bone resorption through a reduction of TNF-alpha production in vivo. However, it was not known how bLF inhibits LPS-mediated TNF-alpha and RANKL (receptor activator of nuclear factor kappa B ligand) production in osteoblasts. In this study we show that bLF impairs LPS-mediated TNF-alpha and RANKL production. bLF inhibited LPS-mediated osteoclastogenesis via osteoblasts in a co-culture system. Furthermore, bLF pretreatment inhibited LPS-induced NF kappa B DNA binding activity as well as I kappa B alpha and IKK beta (I kappa B kinase beta) phosphorylation. MAP kinase activation was also inhibited by bLF pretreatment. However, bLF pretreatment failed to block the degradation of IRAK1 (interleukin-1 receptor-associated kinase 1), which is an essential event after its activation. Remarkably, we found that bLF pretreatment inhibited LPS-mediated Lys-63-linked polyubiquitination of TNF receptor-associated factor 6 (TRAF6). We also found that bLF is mainly endocytosed through LRP1 (lipoprotein receptor-related protein-1) and intracellular distributed bLF binds to endogenous TRAF6. In addition, bLF inhibited IL-1 beta- and flagellin-induced TRAF6-dependent activation of the NF kappa B signaling pathway. Collectively, our findings demonstrate that bLF inhibits NF kappa B and MAP kinase activation, which play critical roles in chronic inflammatory disease by interfering with the TRAF6 polyubiquitination process. Thus, bLF could be a potent therapeutic agent for inflammatory diseases associated with bone destruction, such as periodontitis and rheumatoid arthritis.