Mechanosensing Function of Primary Cilium-Polycysin Complex in Bone

描述（由申请人提供）：机械应变是维持骨量的重要刺激，但负责机械传感的近端分子靶点尚不清楚。本申请的目的是测试新的假设，即初级纤毛-多囊蛋白复合物在出生后骨的成骨细胞和骨细胞中作为机械传感器发挥作用。我们已经发现，多囊蛋白1（PC 1），由PKD 1编码的细胞表面跨膜受体，多囊蛋白2（PC 2），由PKD 2编码的钙通道，和初级纤毛，一个单一的，非运动的，膜覆盖的细胞表面突起，存在并共定位于成骨细胞/骨细胞。基于已知的初级纤毛、PC 1和PC 2组装成机械感应复合物的能力，以及我们的初步研究表明，PC 1突变小鼠在体内和体外对机械负荷的骨和成骨细胞反应受损，并且骨钙素-Cre介导的PKD 1从骨中的选择性缺失导致成年小鼠骨质减少，我们认为初级纤毛/多囊蛋白复合物是成骨细胞和骨细胞中的关键力学传感器。为了进一步确定初级纤毛和PC 1/PC 2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来创建仅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。具体而言，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（DMP 1）启动子驱动的Cre小鼠与floxed PKD 1和KIF 3A小鼠杂交，实现PKD 1和初级纤毛的成骨细胞/骨细胞特异性失活。通过研究这些动物的反应，在体内的机械加载和卸载和成骨细胞和骨细胞来自这些小鼠在体外的机械应变的反应，我们将阐明的mechanosensing作用的初级纤毛和PKD 1在骨和识别信号通路连接这个mechanosensing复合物合成代谢反应。这些研究将确定在成骨细胞和骨细胞中的初级纤毛和多囊蛋白的功能，并将有助于更好地理解机械负荷诱导骨形成的分子机制。公共卫生相关性：这些研究的积极成果将预示着骨生物学研究的一个新领域，这将从根本上影响我们对骨如何感知机械负荷的理解，并将为预防由于固定和微重力引起的骨丢失提供新的见解。通过靶向骨中的纤毛/多囊蛋白，可以开发增加骨量的药理学方法，从而为骨质减少性疾病提供新的治疗方法。

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.