Signaling Mechanisms Of Bovine Lactoferrin-induced Osteoblast Differentiation

Lactoferrin (LF), a multifunctional iron-binding glycoprotein, has been proved as a potent anabolic effector for bone health. However, the molecular mechanisms underlying the effect of lactoferrin are still largely unknown. Here, the signaling pathways that mediate the beneficial effect of lactoferrin on osteoblast differentiation were studied for the first time.In both primary osteoblast and preosteoblast MC3T3-E1, lactoferrin promoted alkaline phosphatase (ALP) activity, osteocalcin (OCN) secretion as well as mineralization, suggesting that lactoferrin could stimulate both early and late differentiation of osteoblast. Meanwhile, lactoferrin also promoted the phosphorylation of transcription factor Runx2which is the key regulator of bone formation. Along with this enhanced osteogenic differentiation, activation of p38mitogen-activated protein kinase was detected in LF-treated MC3T3-E1cells. Down-regulating p38with selective inhibitor SB203580or p38a siRNA attenuated the effect of lactoferrin on osteogenesis. Furthermore, knockdown of p38a significantly decreased LF-induced Runx2phosphorylation. These results showed that LF promoted osteoblast differentiation via p38-Runx2activation. Besides p38MAPK activation, protein kinase A (PKA) was also activated in MC3T3-E1cells. PKA inhibitor H89significantly inhibited LF-induced p38activation, ALP activity and OCN secretion, indicating that PKA possibly acted as an upstream kinase of p38.In order to further identify the role of lactoferrin’s receptor low-density lipoprotein receptor-related protein1(LRP1), we constructed LRP1stable-knockdown MC3T3-E1cells by lentiviral-mediated transfection of pGLV3-LRP1-shRNA vector. LRP1stable-knockdown did not attenuate the LF-induced osteogenesis, implying that lactoferrin stimulated osteoblast differentiation via LRP1-independent pathway. Moreover, we also confirmed the contribution of endocytosis process to the anabolic effect of lactoferrin on osteoblast differentiation. In this study, endocytosis of lactoferrin was significantly blocked by incubating the cells at4degree or culturing in hypertonic medium. Under these conditions, lactoferrin still activated p38MAPK and CREB, which suggested that endocytosis of lactoferrin was not required for the activation of p38MAPK and PKA and other potential lactoferrin receptors probably existed.Finally, we performed an antibody array to analyze the growth factor production in normal and LRP1-knockdown osteoblast with or without lactoferrin stimulation. We detected53growth factors in lactoferrin-treated osteoblast, and the results showed that Thrombospondin-2, Serpin E1, MCP-1, PDGF-AA, Angiogenin, FGF and Cyr61were up-regulated; while Serpin Fl were down-regulated. LF may indirectly regulate osteoblast activity through growth factors.Taken together, the present work indicated that lactoferrin stimulated MC3T3-E1preosteoblast differentiation mainly through LRP1-independent PKA-p38-Runx2signaling pathways. These results provided the first evidence of the signaling mechanisms of lactoferrin’s effect on osteoblast differentiation.