Ultracentrifuged serum fractions reveal promotion of dental epithelial proliferation by lactoferrin

OBJECTIVES: Serum is an essential supplement of basal cell culture media, providing growth factors, adhesion molecules, hormones, lipids, and minerals that support cell survival and function. This study was conducted to investigate the effects of ultracentrifugation-separated fractions of fetal bovine serum (FBS) on the proliferation and differentiation of dental epithelial cells with the aim of identifying the key serum-derived factors that regulate cellular activities. METHODS: FBS was fractionated by ultracentrifugation at 100,000×g for 18 h at 4 °C into three layers, designated as the FBS-1st (top), -2nd (middle), and 3rd (bottom) fractions. Each fraction was added at 10 % (v/v) to the culture medium of rat dental epithelial HAT-7 cells. Cell proliferation was assessed by cell counting and 5-bromo-2'-deoxyuridine incorporation, and differentiation was evaluated by ameloblastin immunostaining. The involvement of lactoferrin and its receptors was examined using recombinant protein stimulation, reverse transcription polymerase chain reaction, small interfering RNA knockdown, and western blotting. RESULTS: FBS-3rd significantly promoted cell proliferation, whereas FBS-1st and -2nd inhibited it. Ameloblastin staining revealed no significant differences between FBS-1st and the control; however, it was suppressed in the FBS-2nd and -3rd. Proteomic analysis identified lactoferrin as the most abundant protein in FBS-3rd, and exogenous lactoferrin enhanced HAT-7 cell proliferation. The lactoferrin receptor, low-density lipoprotein receptor-related protein 1 (Lrp1), was expressed in HAT-7 cells, and Lrp1 knockdown stopped lactoferrin-induced proliferation. Lactoferrin induced phosphorylation of extracellular signal-regulated kinase 1/2, but not Akt serine/threonine kinase, and mitogen-activated protein kinase kinase inhibition with U0126 suppressed the proliferative effect of lactoferrin. CONCLUSION: Lactoferrin, which was enriched in the FBS-3rd fraction, may contribute to the proliferation of dental epithelial cells via the Lrp1-ERK signaling pathway.