机械应力通过Cofilin/NF-κB通路促进成骨

The bone is a very sophisticated tissue which can regulate its mass and architecture to adapt to functional environment. Application of mechanical load promotes bone formation, whereas removal of load leads to bone loss. As osteoblast is highly sensitive to mechanical stimuli, it plays the leading role in the bone remodeling process. However, fundamental cellular and molecular mechanisms of mechanical strain-induced new bone formation are not fully understood. .In our previous work, we identified some differentially expressed proteins by comparing the proteomes of human osteoblast-like Saos-2 cells before and after mechanical strain loading. Among these proteins, Cofilin in the strained group showed statistically significant change compared with controls samples. Recent studies demonstrated that overexpression of wild type Cofilin, known to destabilize the actin cytoskeleton, could impair thrombin-induced NF-κB activity through inhibition of RelA/p65 nuclear translocation in endothelial cells. And it has been shown that NF-κB signal transduction not only promotes osteoclast activation and bone resorption but also simultaneously inhibits osteoblast function, thus limiting the compensatory bone formation that maintains bone homeostasis. Based on the above mentioned evidence, we hypothesize that Cofilin/NF-κB plays essential role in regulating mechanical strain-induced osteoblastic bone formation in osteoblasts..In this project, we will investigate the effects of mechanical strain on the Cofilin/NF-κB signal transduction pathway in Saos-2 osteoblastic cells by using laser scanning confocal microscope, gene transfection, RNAi, Western Blot, MicroCT and other biological techniques. Furthermore, we will evaluate the relationship between the expression of Cofilin and new bone formation in vivo in our previously established animal model of mandibular distraction osteogenesis. .In our opinion, this project will help us to uncover the functions of Cofilin/NF-κB pathway and how they are regulated by medical strain. And the results could be translated into effective disease therapies by considering Cofilin as a novel drugable target in promoting osteogenesis.

力学刺激是调节骨代谢的重要因素，探索成骨细胞的机械生物信号转导机制有助于阐明骨改建的机理和发现调控骨生长的新靶点。在前期工作中，通过对应力加载前后成骨细胞的差异蛋白质组学分析，我们发现应力干预能够显著上调细胞骨架调节蛋白Cofilin的表达。结合文献，我们提出Cofilin表达上调导致NF-κB抑制是机械应力促进成骨的新机制。本项目拟以机械应力干预的成骨细胞和下颌牵张成骨动物分别作为体外和在体研究对象，使用激光共聚焦、RNAi、基因转染、Western Blot、MicroCT等研究手段，系统探讨机械应力作用下成骨细胞内Cofilin表达和NF-κB活性与细胞成骨特性之间的关系，证实机械应力通过上调Cofilin表达来抑制NF-κB活性通路促进成骨，从而丰富成骨细胞的机械生物信号转导网络，同时为缩短牵张成骨时程、加速骨缺损修复开辟新的思路，并为筛选治疗骨代谢失衡相关疾病的药物提供新靶点。

正常骨骼处于一个吸收与重建的动态平衡状态中，其中力刺激是维持平衡的基础。不断有研究证实，牵张应力是有效促进成骨活性和功能的力学刺激。体内外力刺激通过多种途径被成骨细胞所感知，通过一系列的级联网络反应，成骨细胞将力学信号转化为生物学信号，进一步调节相关基因的表达水平，同时合成各种活性物质及酶类，最终使得机体表现出各种复杂的生理及病理活动。多种信号转导途径中，细胞骨架作为首先感受力学刺激的结构，发挥着重要作用。成骨细胞对外源性机械力信号的反应主要是由F-actin来响应的，F-actin通过与跨膜分子相互作用而成为跨膜力信号传递的主要环节。而成骨细胞细胞骨架的改建依赖于一些对力学敏感的关键蛋白的调控作用。ADF/Cofilin家族蛋白是actin结合蛋白中独一无二的解聚微丝的蛋白群，由LIM激酶LIMK1介导的对Cofilin磷酸化抑制了Cofilin解聚肌动蛋白的能力，Rho蛋白又位于这一通路上游对细胞骨架起一定的调节作用。 进而推断在细胞受到外力刺激时，Rho蛋白以及Cofilin在细胞骨架介导的信号传导过程中发挥着独特的作用。.本研究通过Flexcell应力加载系统，对人成骨样细胞（MG-63）施以不同加载时长的周期性牵张力，通过免疫荧光、RT-PCR、Western Blot、基因沉默等技术，探讨MG-63细胞受牵张力刺激后Cofilin的表达情况，以及在Cofilin正常表达与Cofilin抑制后，对上下游的成骨目的基因的影响，从而探讨Cofilin在机械力学刺激下细胞信号转导中的作用。在Cofilin的变化明确后对MG-63进行12%力学加载，通过RT-PCR、Western-blot等方法，观察Rho蛋白和下游效应分子ROCK以及Cofilin表达之间的联系，从而对Rho/Rock/Cofilin信号通路产生更多的认识，另外通过对RhoA蛋白进行抑制，观察Rho/Rock通路对成骨效应影响，进而深入地了解成骨细胞信号传导的途径。为骨力学转导机制提供新的依据，为缩短牵张成骨时程、加速骨缺损修复开辟新的思路，并为筛选治疗骨代谢失衡相关疾病的药物提供新靶点。