Molecular Mechanisms Of Lactoferrin-induced Osteogenesis Through TGF-? Receptor And VD Receptor

Lactoferrin,as a promising anabolic factor for bone health,has been widely used as nutritional supplement in infant dairy products.However,the molecular mechanisms underlying the osteotropic activity of LF are still largely unknown.In this study,we firstly clarified TGF-? receptor ?(T?R?)as the receptor of LF on osteoblast proliferation and differentiation.In addition,LF also increased bone mineral density and the structure of trabecular.The study could be providing a new evidence for the osteotropic activity of LF and a theoretical basis for the precise design of infant dairy products.Co-immunoprecipitation was used to illuminate the interaction of LF and T?R?.We verified that T?R? and T?R? were required for LF-induced osteoblast proliferation and differentiation using lentiviral-mediated vector and inhibitor SB431542.Futhermore,we demonstrated that LF activated p38 MAPK independented on TGF-? receptor.To investigate the effect of Vitamin D receptor(VDR)on LF-induced osteoblast proliferation and differentiation.The expression of VDR was increased significantly after LF treatment.Down-regulating VDR with siRNA attenuated the effect of lactoferrin on osteogenesis.In addition,the expression of VDR was still increased by LF when the presence of 1,25-dihydroxyvitamin D3.These results showed that LF promoted osteoblast proliferation and differentiation via VDR activation.In order to further identify the role of VDR,4-month-old male C57BL/6J mice were orally intubated with LF at 100 and 1000mg/kg body weight.The bone metabolism biochemical markers were detected in blood and urine.These findings indicated that oral LF has no significant effect on calcium and phosphorus in blood,except for the markers in urine.Besides,not only bone mass but also bone microarchitecture were increased in LF-fed mice at all doses.Moreover,we detected the expression of VDR in kidney,colon,and femur tissue,and the results showed that VDR was an important pathway for the osteogenic activity of LF.To investigate the mechanism of LF-induced the expression of VDR.,down-regulating p38 or TGF-?/smads with selective inhibitors did not attenuate the expression of VDR induced by LF.The results indicated that LF induced the expression of VDR independented on signaling pathway.In addition,endocytosis of LF was significantly blocked by incubating the cells at 4 degree or culturing in hypertonic medium.Under these conditions,LF could not induce the expression of VDR,which suggested that the site of the action of LF is intracellular.Taken together,the present work firstly indicated the interaction between LF and T?R?.TGF-?receptors were required for LF-induced osteoblast proliferation and differentiation in MC3T3-E1 cells.LF,as a VDR agonist,regulated the osteogenic activity.These findings indicated that oral LF not only preserved bone mass but also improved bone microarchitecture via the activation of VDR.