Mechanosensing function of primary cilium-polycysin complex in bone

描述(申请人提供)：机械应变是维持骨量的重要刺激因素，但负责机械感应的最近的分子靶点尚不清楚。这项应用的目的是检验这一新的假设，即初级纤毛-多囊蛋白复合体作为机械传感器在成骨细胞和出生后骨细胞中发挥作用。我们发现在成骨细胞/骨细胞中存在由PKD1编码的细胞表面跨膜受体多囊蛋白1(PC1)，由PKD2编码的钙通道多囊蛋白2(PC2)，以及单个的、不运动的、有膜覆盖的细胞表面突起的初级纤毛。根据已知的初级纤毛、PC1和PC2组装成机械传感复合体的能力，以及我们的初步研究表明，PC1突变小鼠在体内和体外对机械载荷的骨和成骨细胞反应受损，骨钙素-Cre介导的PKD1从骨中选择性缺失导致成年小鼠骨量减少，我们认为初级纤毛/多囊蛋白复合体是成骨细胞和骨细胞中关键的机械传感器。为了进一步确定初级纤毛和PC1/PC2复合体在成骨细胞谱系中的重要性，我们将使用小鼠遗传学方法建立只在成骨细胞和骨细胞中选择性缺乏初级纤毛和多囊蛋白的动物模型。具体地说，我们将使用CRE条件(LOX-P)系统，通过将骨钙素和牙本质基质蛋白1(DMP1)启动子驱动的CRE小鼠与PKD1和KIF3A小鼠杂交，实现成骨细胞/骨细胞特异性的PKD1和初级纤毛的灭活。通过检测这些动物在体内对机械加载和卸载的反应以及这些小鼠来源的成骨细胞和骨细胞对机械应变的反应，我们将阐明初级纤毛和PKD1在骨骼中的机械传感作用，并确定将这种机械传感复合体与合成代谢反应联系起来的信号通路。这些研究将确定初级纤毛和多囊蛋白在成骨细胞和骨细胞中的功能，并有助于更好地理解机械载荷诱导骨形成的分子机制。公共卫生相关性：这些研究的积极结果将预示着骨生物学研究的一个新的研究领域，这将从根本上影响我们对骨骼如何感知机械负荷的理解，并将为预防由于固定和微重力造成的骨丢失提供新的见解。通过靶向骨骼中的纤毛/多囊蛋白，可以开发增加骨量的药理学方法，从而为骨量减少症的新治疗提供潜在的可能性。

DESCRIPTION (provided by applicant): Mechanical strain is an important stimulus for maintaining bone mass, but the proximate molecular target responsible for mechanosensing is not known. The objective of this application is to test the novel hypothesis that the primary cilium-polycystin complex functions as a mechanosensor in osteoblasts and osteocytes in postnatal bone. We have discovered that polycystin 1 (PC1), a cell-surface transmembrane receptor encoded by PKD1, polycystin 2 (PC2), a calcium channel encoded by PKD2, and primary cilium, a single, non-motile, membrane-covered cell surface projection, are present and co-localize in osteoblasts/osteocytes. Based on the known ability of primary cilium, PC1 and PC2 to assemble into a mechanosensing complex and our preliminary studies showing that PC1 mutant mice have impaired bone and osteoblastic response to mechanical loading in vivo and in vitro and that the Osteocalcin-Cre mediated selective deletion of PKD1 from bone results in osteopenia in adult mice, we propose that the primary cilium/polycystin complex is a key mechanosensor in osteoblasts and osteocytes. To further establish the importance of primary cilium and the PC1/PC2 complex in the osteoblast lineage, we will use mouse genetic approaches to create animal models that selectively lack primary cilium and polycystins only in osteoblasts and osteocytes. Specifically, we will use the Cre-conditional (lox-P) system to achieve osteoblast/osteocyte-specific inactivation of PKD1 and primary cilium by crossing Osteocalcin and Dentin Matrix Protein 1 (DMP1) promoter driven Cre mice with floxed PKD1 and KIF3A mice. By examining the response of these animals to mechanical loading and unloading in vivo and the response of osteoblasts and osteocytes derived from these mice to mechanical strain in vitro, we will elucidate the mechanosensing role of primary cilium and PKD1 in bone and identify signaling pathways linking this mechanosensing complex to anabolic responses. These studies will define the function of primary cilium and polycystins in osteoblasts and osteocytes and will contribute to a better understanding of molecular mechanisms underlying mechanical load-induced bone formation. PUBLIC HEALTH RELEVANCE: The positive outcome of these investigations will herald a new area of investigation in bone biology research that will impact fundamentally on our understanding of how bone senses mechanical loading and will provide new insights into prevention of bone loss due to immobilization and microgravity. Development of pharmacological approaches to increased bone mass might be developed by targeting cilia/polycystins in bone, thereby offering the potential of new treatments for osteopenic disorders.