Lactoferrin Stimulates Osteoblast Differentiation Through PKA and p38 Pathways Independent of Lactoferrin's Receptor LRP1
Lactoferrin Stimulates Osteoblast Differentiation Through PKA and p38 Pathways Independent of Lactoferrin's Receptor LRP1
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DOI：
10.1002/jbmr.2116
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发表时间：
2014-05-01
影响因子：
6.2
通讯作者：
Ren, Fazheng
中科院分区：
文献类型：
--
作者：
Zhang, Wei;Guo, Huiyuan;Ren, Fazheng
乳铁蛋白（LF）已被确定为体内和体外骨骼健康的有效合成代谢因子。然而，LF作用的分子机制仍然是未知的。在这里，我们探讨了介导LF对成骨细胞分化的有益作用的信号通路。在原代成骨细胞和前成骨细胞MC 3 T3-E1中，LF促进碱性磷酸酶（ALP）活性、骨钙素（OCN）分泌和矿化。沿着这种增强的成骨分化，在LF处理的MC 3 T3-E1细胞中检测到p38丝裂原活化蛋白激酶（MAPK）的活化。用选择性抑制剂SB 203580或p38小干扰RNA（siRNA）下调p38可减弱LF对成骨的影响。此外，敲低p38显著降低LF诱导的Runt相关转录因子2（Runx 2）磷酸化。根据以往的研究和我们的结果，我们推测LF诱导的成骨细胞增殖和分化是两个相对独立的过程，分别由细胞外信号调节激酶1/2（ERK 1/2）和p38通路控制。在MC 3 T3-E1细胞中，除了p38 MAPK激活外，蛋白激酶A（PKA）也被激活。PKA抑制剂H89显著抑制LF诱导的p38激活、ALP活性和OCN分泌，表明PKA可能作为p38的上游激酶。为了进一步研究LF受体低密度脂蛋白受体相关蛋白1（LRP 1）的作用，我们构建了LRP 1稳定敲低的MC 3 T3-E1细胞。无论是LRP 1拮抗剂受体相关蛋白（RAP），也没有LRP 1敲低的方法可以减弱LF诱导的成骨，这意味着LF刺激成骨细胞分化通过LRP 1非依赖性途径。综上所述，本工作表明LF主要通过LRP 1非依赖性PKA和p38信号通路促进MC 3 T3-E1前成骨细胞分化。这些结果首次为LF影响成骨细胞分化的信号转导机制提供了证据。(c)2014年美国骨与矿物质研究学会。
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 p38 small interfering RNA (siRNA) attenuated the effect of LF on osteogenesis. Furthermore, knockdown of p38 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. (c) 2014 American Society for Bone and Mineral Research.