骨细胞自噬在正畸牙移动中的作用及其分子机制

Orthodontic tooth movement (OTM) occurs as a result of alveolar bone remodeling in response to applied force that produces two different regions around the tooth: compression and tension. On the compression side, there is osteolysis followed by the activation of osteoclasts；on the tension side, there is new bone formation followed by the activation of osteoblasts. So far, the tissue, cellular, and molecular regulation of alveolar remodeling during OTM is still complicated. Osteocytes are considered as major mechanosensory cells of the bone which can transduce the mechanical signals into biological cues. Osteocytes can also regulate the function of neighboring cells through gap junctions and (or) secreting various growth factors. Given these studies, there is an increasing interest in the role of osteocytes in regulating alveolar bone remodeling during OTM. Now the underlying mechanism how osteocytes regulate osteoblasts/osteoclasts during OTM is becoming the emphasis and difficulty in orthodontic filed. Autophagy is an intracellular recycling pathway in which cellular components, including protein aggregates and organelles, are targeted to the lysosome for degradation. Autophagy is involved in various biological processes, including maintaining bone homeostasis and regulating bone remodeling. ATG7, as a member of the ATG protein family, is involved in the occurrence of autophagy and can be targeted to modulate the process. Previous study demonstrated that deletion of a conditional allele for Atg7 from osteocytes caused suppression autophagy in osteocytes and decrease in bone mass, cortical thickness, osteoclast number and osteoblast number, suggesting that osteocyte autophagy is essential to bone homeostasis. But whether osteocyte autophagy occurs and how it affects the alveolar bone remodeling during OTM remains unkown. Our previous results showed: In-vitro tension could promote autophagy in osteolike cell MLO-Y4, accompanied by the increased ATG7 and upstream CARM1/AMPK. Cocultured with the MLO-Y4 under strain loading, proliferation and osteogenic differentiation of osteoblast (MC3T3E1) increased. In-vivo mouse OTM model suggested that orthodontic force could upregulate osteocyte autophagy. Therefore, we hypothesis that the AMPK-CARM1-ATG7 signaling pathway regulating the osteocyte autophagy modifies the bone remodeling. In order to prove our hypothesis, the study was designed into two parts (in-vitro and in-vivo). In vitro, two coculture cell models were constructed: (i) osteocyte-osteoclast under compression stimulation condition model, (ii) and osteocyte-osteoblast under strain stimulation condition model. Then the occurrence of osteocyte autophagy, the function of osteoclasts and the proliferation/differentiation of osteoblasts will be tested. To further elucidate the mechanism how osteocytes regulate the osteoclasts and osteoblasts, the expression of paracrine factors (RANKL/OPG et al.) from osteocyte will be detected. To investigate the function of AMPK-CARM1-ATG7 signaling pathway in mechanical stress induced osteocyte autophagy, we plan to down-regulate the expression of AMPK/CARM1/ATG7 respectively using lenti-virus infection technology and up-regulate AMPK with its agonist (Acadesine). In vivo, conditional knock-out Dmp1-Cre;Atg7-flox/ flox mice OTM model will be generated , and WT mice treated with the AMPK inhibitor(Compound C,IV) or agonist (Acadesine,IP) OTM model will be used as well. Then the activation of the signaling pathway, the occurrence of the osteocyte autophagy, and the bone remodeling will be examined. In summary, the project is to reveal the mechanism that orthodontic applied force could activate the AMPK-CARM1-ATG7 signaling pathway, then regulate the level of osteocyte autophagy, and finally influence the function of osteoblasts and osteoclasts during OTM. Understanding this mechanism may suggest a potential solution for OTM acceleration in future.

骨细胞感受机械应力，活化破骨细胞和成骨细胞，是正畸牙移动牙槽骨改建的中心环节之一，其分子机制不清。骨细胞自噬是骨组织发生改建的一种新的调控机制。前期试验观察到骨细胞自噬与正畸牙移动相关，但其如何参与牙槽骨改建的作用机制尚未明了。本课题拟研究骨细胞自噬AMPK-CARM1-ATG7信号通路在正畸牙移动中的作用。体外，构建机械力加载的骨细胞-破骨细胞/骨细胞-成骨细胞三维共培养模型，通过上调或下调AMPK/CARM1/ATG7信号分子，检测对骨细胞自噬的影响，探讨干预骨细胞自噬对破骨细胞、成骨细胞功能的影响；体内，运用小鼠牙移动模型，通过注射AMPK抑制剂/激动剂，以及构建Dmp1-Cre;Atg7-flox/flox模式小鼠，观察调控AMPK-CARM1-ATG7信号通路对正畸牙移动的影响，明确骨细胞自噬在正畸牙移动中的作用。这将为临床探索以骨细胞自噬为靶点来加速牙移动的方法提供理论依据。

正畸牙移动是由机械力驱动的，包括机械张力诱导的骨生成和机械压力诱导的骨吸收。了解其中的机制，可为加速正畸牙移动提供临床依据。骨细胞是机械敏感细胞，可以通过感受机械刺激来调控骨改建。自噬是细胞内一个高度保守的过程，在各种外界刺激下被激活。在本研究中，我们假设在正畸牙移动过程中，骨细胞的自噬被机械力激活，从而促进牙槽骨的改建以及牙齿移动。我们发现与牙移动组小鼠与对照组相比，牙槽骨张力侧及压力侧骨细胞自噬水平升高。体外实验发现，骨细胞可通过感受压应力或张应力而激活自噬，随后分别分泌RANKL促进破骨活动及分泌FGF23促进成骨活动。我们初步探究了机械力促进骨细胞自噬的信号通路，发现ROCK-AKT-TFE3及AMPK信号通路在其中发挥作用。最后，我们进一步构建Dmp1Cre ERT2;Atg7fl/fl小鼠，在骨细胞中特异性敲除Atg7基因。研究结果表明，敲基因组牙移动距离均显著小于对照组。且敲基因组压力侧破骨活性及张力侧成骨活性均低于对照组，提示骨细胞的自噬在促进骨改建及正畸牙移动中发挥重要作用。