Significance of the neuronal guidance molecule Semaphorin 3A for bone remodelling during orthodontic tooth movement

在学龄儿童和成人中，错颌和下颌错位的发病率很高。除多种细胞因子和生长因子外，神经引导分子（neural guidance molecules， NGM）因其在骨稳态中的作用以及在牙齿运动中的潜在作用而受到关注。最近我们已经证明Ephrin配体和Eph受体参与了牙齿运动过程中的骨重塑。在最近发现的与骨重塑调控有关的ngm中，信号蛋白3A （Sema3A）是最有趣的，因为它同时诱导成骨细胞的成骨分化和干扰破骨细胞的分化。为了继续分析正畸牙齿运动中的神经元引导分子并阐明其潜在机制，本项目的目标是：(1)研究不同的机械力是否会调节牙周成纤维细胞和牙槽骨成骨细胞中Sema3A及其受体Neuropilin1和PlexinA1的表达；（ii）研究哪些生化信号参与了Sema3A的机械调节；（iii）研究Sema3A刺激对肺泡骨成骨细胞分化的影响；（iv）阐明Sema3A依赖性成骨细胞分化的分子机制。为了达到这个目的，我将首先通过施加压缩和张力来机械地刺激牙周成纤维细胞和牙槽骨成骨细胞。两种细胞类型的培养以及机械刺激在我们的实验室已经很好地建立起来了。采用定量PCR和western blot方法分析Sema3A及其受体的表达。其次，我将研究导致Sema3A改变的潜在机械诱导途径。为此，我们测试了不同的map激酶的磷酸化情况，并将通过qPCR和western blot研究假定的Sema3A转录因子Osterix的参与。第三，重组人Sema3A刺激肺泡成骨细胞，通过定量PCR监测不同成骨细胞分化标志物的表达，分析其对成骨分化的影响。第四，通过拉下实验、免疫荧光染色和western blot分别研究Rac1 GTPase和β -catenin转录因子的参与，探讨Sema3A介导成骨细胞分化的机制。我们对Ephrin/Eph家族成员的研究和Sema3A的初步数据表明，神经元引导分子在正畸牙齿运动调节中的作用可能被低估了。因此，该项目可能阐明额外的细胞和分子事件，这些事件可能有用的药理学操作目标，旨在实现无副作用和加速正畸牙齿运动。

Malocclusions and jaw misalignments appear with high prevalence in school children and in adults. Besides various cytokines and growth factors neuronal guidance molecules (NGM) gained attention for their roles in bone homeostasis and thus potential roles during tooth movement. Recently we have shown that Ephrin ligands and Eph receptors are involved in bone remodeling during tooth movement. Among the NGMs which have recently been implicated in the regulation of bone remodeling Semaphorin 3A (Sema3A) is the most interesting as it concurrently induces osteogenic differentiation in osteoblasts and disturbs osteoclast differentiation. To continue my analysis of neuronal guidance molecules in orthodontic tooth movement and to elucidate the underlying mechanisms the objectives of this project are (i.) to investigate if different mechanical forces modulate the expression of Sema3A and its receptors Neuropilin1 and PlexinA1 in periodontal fibroblasts and osteoblasts of the alveolar bone, (ii.) to study which biochemical signals are involved in the mechanical modulation of Sema3A (iii.) to investigate the effects of Sema3A stimulation on alveolar bone osteoblasts differentiation and (iv.) to elucidate the molecular mechanisms underlying the Sema3A dependent osteoblast differentiation. To achieve this, I will first stimulate periodontal fibroblasts and alveolar bone osteoblasts mechanically by applying compressive and tension forces. Culture of both cell types, as well as the mechanical stimulation is well established in our laboratory. The expression of Sema3A and its receptors will be analyzed by means of quantitative PCR and western blot. Second, I will investigate the potential mechanoinductive pathway leading to Sema3A alterations. To this end, different MAPKinases are tested for phosphorylation and the involvement of the putative Sema3A transcription factor Osterix will be studied by qPCR and western blot. Third, alveolar osteoblasts will be stimulated by recombinant human Sema3A and its impact on osteogenic differentiation will be analyzed by monitoring the expression of different osteoblast differentiation markers by quantitative PCR. Fourth, the mechanism underlying the Sema3A mediated osteoblast differentiation will be investigated by studying the involvement of the Rac1 GTPase and the beta-catenin transcription factor using pull down assays and immunofluorescent staining and western blot, respectively. Own research on members of the Ephrin/Eph family and preliminary data on Sema3A suggest that the role of neuronal guidance molecules in the regulation of orthodontic tooth movement might have been underestimated. Thus, this project might illuminate additional cellular and molecular events which might be useful as targets for pharmacological manipulations aiming for a side effect free and accelerated orthodontic tooth movement.