Lactoferrin promotes osteogenesis through TGF-β receptor II binding in osteoblasts and activation of canonical TGF-β signaling in MC3T3-E1 cells and C57BL/6J mice

Background: Lactoferrin (LF), as a major functional protein in dairy products, is known to modulate bone anabolic effects. However, the underlying molecular mechanisms remain unclear; the receptor of LF in osteoblast differentiation has not been identified. Objective: The aims of the study were to 1) illuminate whether the receptor of LF in osteoblast differentiation is transforming growth factor-β (TGF-β) receptor (TβR) II and 2) determine whether the TGF-β signaling pathway is activated by LF in promoting osteogenesis in vitro and in vivo, in addition to P38 and extracellular signal-regulated kinase (ERK) pathways. Methods: We utilized co-immunoprecipitation to detect any binding of LF to TβRII. Subsequently, the role of the TGF-β signaling pathway involved in LF-induced osteoblast proliferation and differentiation was determined by inhibition of TβRI activity by inhibition and knockout of TβRII expression by small guide RNA (sgRNAs) in MC3T3-E1 cells. In addition, 4-wk-old male C57BL/6J mice were orally administered 100 mg LF/kg body weight for 16 wk, after which any activation of the TGF-β signaling pathway in vivo was measured by Western blots. Results: LF was shown to directly interact with the TβRII protein and to activate the TGF-β signaling pathway in MC3T3-E1 cells. Inhibition of TβRI activity and knockout TβRII expression both attenuated the stimulation of LF in osteoblast proliferation and differentiation by 30-50%. LF-induced activation of TGF-β canonical signaling resulted in upregulation of osteogenic factors. Moreover, the expression of phosphorylated-drosophila mothers against decapentaplegic protein 2 (SMAD2) was increased by 1-fold after LF treatment in the femoral tissue of mice. Conclusions: This study provides evidence identifying TβRII as an LF receptor in LF-induced osteoblast differentiation. In addition, the TβRII-dependent TGF-β canonical signaling pathways were proven to play an important role in mediating LF-induced osteogenesis both in MC3T3-E1 cells and in C57BL/6J mice.