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

Toshihiro Inubushi; Aki Kawazoe; Mutsumi Miyauchi; Yasusei Kudo; Min Ao; Atsushi Ishikado; Taketoshi Makino; Takashi Takata

doi:10.1074/jbc.M111.324673

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

From the ^‡Department of Oral and Maxillofacial Pathobiology,
^§Department of Orthodontics and Craniofacial Developmental Biology, and
^¶Department of Pediatric Dentistry, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima 734-8553, Japan and
^‖Health Care R&D Division, Sunstar, Osaka 569-1195, Japan

↵1 To whom correspondence may be addressed: Dept of Oral and Maxillofacial Pathobiology, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. Tel.: 81-82-257-5634; Fax: 81-82-257-5619; E-mail: mmiya{at}hiroshima-u.ac.jp.
↵2 To whom correspondence may be addressed: Dept. of Oral and Maxillofacial Pathobiology, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan. Tel.: 81-82-257-5634; Fax: 81-82-257-5619; E-mail: ttakata{at}hiroshima-u.ac.jp.

Background: We previously reported that bovine lactoferrin (bLF) reduces LPS-induced bone resorption. However, it was not clear how bLF inhibits LPS-mediated osteoclastogenesis through osteoblasts.

Results: bLF impaired LPS-mediated NFκB activation by interfering with TNF receptor-associated factor 6 (TRAF6) polyubiquitination.

Conclusion: bLF is a potent anti-inflammatory agent.

Significance: bLF may hold promise as a therapeutic agent for inflammatory diseases associated with bone destruction.

Abstract

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-α production in vivo. However, it was not known how bLF inhibits LPS-mediated TNF-α and RANKL (receptor activator of nuclear factor κB ligand) production in osteoblasts. In this study we show that bLF impairs LPS-mediated TNF-α and RANKL production. bLF inhibited LPS-mediated osteoclastogenesis via osteoblasts in a co-culture system. Furthermore, bLF pretreatment inhibited LPS-induced NFκB DNA binding activity as well as IκBα and IKKβ (IκB kinase β) 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-1β- and flagellin-induced TRAF6-dependent activation of the NFκB signaling pathway. Collectively, our findings demonstrate that bLF inhibits NFκB 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.