Modulation of Bone Formation by SHN3

 描述（由申请人提供）：以前，我们发现衔接蛋白SHN 3通过抑制促分裂原活化蛋白激酶（MAPK）ERK的活性，对体内成骨细胞抑制骨形成具有重要作用。我们还发现WNTs具有以前未鉴定的激活SHN 3/ERK通路的活性。在这里，我们试图通过确定SHN 3如何抑制ERK以及该途径如何影响体内骨骼疾病模型来对这些观察结果进行重要的扩展。这一领域的研究是重要的，因为骨质疏松症是一种高度流行的低骨量和骨骼脆弱性疾病。一半的女性在其一生中会经历过腰椎骨折，这些骨折中的每一种都会引起显著的发病率和死亡率。基于SHN 3抑制骨形成的深刻能力，更好地理解这一途径将产生控制成骨细胞形成骨的活性的新方法，可能提供治疗骨质疏松症和其他低骨量疾病的方法。在这个项目中要调查的三个重叠领域是：1。确定SHN 3对骨骼疾病模型的贡献虽然我们已经确定SHN 3在基线时强烈抑制小鼠中的骨形成，但是SHN 3对绝经后骨质疏松症和骨折的鼠模型的具体贡献是未知的，并且将在SHN 3缺陷小鼠中进行研究。2.确定SHN 3如何抑制成骨细胞中的ERK通路虽然我们已经确定SHN 3主要通过抑制ERK的活性在成骨细胞中起作用，但相关的ERK底物以及SHN 3如何塑造ERK响应的动力学尚不清楚，将使用磷酸蛋白质组学和定量ERK活性生物传感器进行研究。确定ERK的相关底物可以鉴定SHN 3/ERK途径的下游效应物，其本身适合于治疗靶向。此外，WNT激活ERK/SHN 3通路的机制尚不清楚，这将通过重点筛选来确定该通路近端激活所需的MAP 3 K。3.确定染色质是否作为支架协调成骨细胞中SHN 3/ERK复合物的活性。SHN 3和ERK之前已经被鉴定为结合DNA，提高了SHN 3调节ERK活性发生在染色质结合复合物中的可能性。在这个目标中，一系列的染色质免疫沉淀测序实验将被用来探索染色质是否协调SHN 3/ERK通路的功能。

 DESCRIPTION (provided by applicant): Previously we found that the adapter protein SHN3 had a major effect to suppress bone formation by osteoblasts in vivo by inhibiting the activity of the mitogen activated protein kinase (MAPK) ERK. We also identified that WNTs have a previously unidentified activity to activate this SHN3/ERK pathway. Here we seek to make critical extensions of these observations by determining how SHN3 inhibits ERK and how this pathway influences models of skeletal disease in vivo. This area of study is significant as osteoporosis is a highly prevalent disorder of low bone mass and skeletal fragility. Half of all women will experience of an osteoporotic fracture during their lifetime, with each of these fractures incurring significant morbidity and mortality. Based on the profound ability of SHN3 to suppress bone formation, building a better understanding of this pathway will yield new methods to control the activity of osteoblasts to form bone, potentially offering approaches to treat osteoporosis and other disorders of low bone mass. The three overlapping areas to be investigated in this project are: 1. Determine the contribution of SHN3 to skeletal disease models while we have established that SHN3 strongly suppresses bone formation in mice at baseline, the specific contribution of SHN3 to murine models of post-menopausal osteoporosis and skeletal fracture are unknown and will be studied in SHN3-deficient mice. 2. Determine how SHN3 inhibits the ERK pathway in osteoblasts While we have established that SHN3 acts in osteoblasts primarily by suppressing the activity of ERK, the relevant ERK substrates and how SHN3 shapes the kinetics of the ERK response are unknown and will be studied using phosphoproteomics and a quantitative ERK activity biosensor. Determining the relevant substrates of ERK may identify downstream effectors of the SHN3/ERK pathway that are themselves amenable to therapeutic targeting. Additionally, the mechanism for the activation of the ERK/SHN3 pathway by WNTs is unknown, and this will be addressed by a focused screen to determine the MAP3K required for proximal activation of this pathway. 3. Determine if chromatin acts as a scaffold to orchestrate the activity of the SHN3/ERK complex in osteoblasts. Both SHN3 and ERK have previously been identified to bind DNA, raising the possibility that regulation of ERK activity by SHN3 occurs in chromatin bound complexes. In this aim, a complimentary series of chromatin immunoprecipitation-sequencing experiments will be utilized to explore if chromatin orchestrates the function of the SHN3/ERK pathway.