Bleedorn Jason A, Hornberger Troy A, Goodman Craig A, Hao Zhengling, Sample Susannah J, Amene Ermias, Markel Mark D, Behan Mary, Muir Peter
Comparative Orthopaedic Research Laboratory, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
PLoS One. 2018 Feb 27;13(2):e0192760. doi: 10.1371/journal.pone.0192760. eCollection 2018.
Mechanical signals play an integral role in the regulation of bone mass and functional adaptation to bone loading. The osteocyte has long been considered the principle mechanosensory cell type in bone, although recent evidence suggests the sensory nervous system may play a role in mechanosensing. The specific signaling pathways responsible for functional adaptation of the skeleton through modeling and remodeling are not clearly defined. In vitro studies suggest involvement of intracellular signaling through mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), and mammalian target of rapamycin (mTOR). However, anabolic signaling responses to bone loading using a whole animal in vivo model have not been studied in detail. Therefore, we examined mechanically-induced signaling events at five time points from 0 to 24 hours after loading using the rat in vivo ulna end-loading model. Western blot analysis of bone for MAPK's, PI3K/Akt, and mTOR signaling, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to estimate gene expression of calcitonin gene-related protein alpha (CGRP-α), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), c-jun, and c-fos in dorsal root ganglion (DRG) of the brachial intumescence were performed. There was a significant increase in signaling through MAPK's including extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) in loaded limbs at 15 minutes after mechanical loading. Ulna loading did not significantly influence expression of the genes of interest in DRG neurons. Bone signaling and DRG gene expression from the loaded and contralateral limbs was correlated (SR>0.40, P<0.05). However, bone signaling did not correlate with expression of the genes of interest in DRG neurons. These results suggest that signaling through the MAPK pathway may be involved in load-induced bone formation in vivo. Further characterization of the molecular events involved in regulation of bone adaptation is needed to understand the timing and impact of loading events, and the contribution of the neuronal signaling to functional adaptation of bone.
机械信号在骨量调节以及骨骼对负荷的功能适应性方面发挥着不可或缺的作用。长期以来，骨细胞一直被视为骨骼中主要的机械感受细胞类型，尽管最近有证据表明感觉神经系统可能在机械感受中发挥作用。通过塑形和重塑实现骨骼功能适应的具体信号通路尚未明确界定。体外研究表明，有丝分裂原激活蛋白激酶（MAPK）、磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（Akt）以及雷帕霉素靶蛋白（mTOR）参与了细胞内信号传导。然而，使用全动物体内模型对骨骼负荷的合成代谢信号反应尚未进行详细研究。因此，我们利用大鼠体内尺骨末端负荷模型，在负荷后0至24小时的五个时间点检查了机械诱导的信号事件。对骨骼进行MAPK、PI3K/Akt和mTOR信号的蛋白质印迹分析，并进行定量逆转录聚合酶链反应（qRT-PCR），以估计臂丛神经节背根神经节（DRG）中降钙素基因相关蛋白α（CGRP-α）、脑源性神经营养因子（BDNF）、神经生长因子（NGF）、c-jun和c-fos的基因表达。机械负荷后15分钟，负荷肢体中包括细胞外信号调节激酶（ERK）和c-Jun氨基末端激酶（JNK）在内的MAPK信号显著增加。尺骨负荷对DRG神经元中相关基因的表达没有显著影响。负荷肢体与对侧肢体的骨信号和DRG基因表达具有相关性（SR>0.40，P<0.05）。然而，骨信号与DRG神经元中相关基因的表达没有相关性。这些结果表明，通过MAPK途径的信号传导可能参与体内负荷诱导的骨形成。需要进一步表征参与骨骼适应性调节的分子事件，以了解负荷事件的时间和影响，以及神经元信号对骨骼功能适应性的贡献。
