Lactoferrin stimulates osteoblast differentiation through PKA and p38 pathways independent of lactoferrin's receptor LRP1.

Lactoferrin (LF) has been established as a potent anabolic factor for bone health both in vivo and in vitro. However, the molecular mechanisms underlying LF's action are still largely unknown. Here, we explore the signaling pathways that mediate LF's beneficial effect on osteoblast differentiation. In primary osteoblast and preosteoblast MC3T3‐E1, LF promoted alkaline phosphatase (ALP)activity, osteocalcin (OCN) secretion, and mineralization. Along with this enhanced osteogenic differentiation, activation of p38 mitogen‐activated protein kinase (MAPK) was detected in LF‐treated MC3T3‐E1 cells. Downregulating p38 with selective inhibitor SB203580 or p38a small interfering RNA (siRNA) attenuated the effect of LF on osteogenesis. Furthermore, knockdown of p38α significantly decreased LF‐induced Runt‐related transcription factor 2 (Runx2) phosphorylation. According to previous studies and our results, we speculated that LF‐induced osteoblast proliferation and differentiation were two relatively separate processes controlled by extracellular signal‐regulated kinase 1/2 (ERK1/2) and p38 pathways, respectively. Besides p38 MAPK activation, protein kinase A(PKA) was also activated in MC3T3‐E1 cells. PKA inhibitor H89 significantly inhibited LF‐induced p38 activation, ALP activity, and OCN secretion, indicating that PKA possibly acted as an upstream kinase of p38. In order to further identify the role of LF's receptor low-density lipoprotein receptor‐related protein 1 (LRP1), we constructed LRP1 stable‐knockdown MC3T3‐E1 cells. Neither LRP1 antagonist receptor associated protein (RAP), nor LRP1 knockdown approach could attenuate the LF‐induced osteogenesis, implying that LF stimulated osteoblast differentiation via an LRP1‐independent pathway. Taken together, the present work indicated that LF stimulated MC3T3‐E1 preosteoblast differentiation mainly through LRP1‐independent PKA and p38 signaling pathways. These results provided the first evidence of the signaling mechanisms of LF's effect on osteoblast differentiation.