Primary Cilia and Bone Homeostasis

原发纤毛和骨稳态

• 批准号: 7918096
• 负责人: Bradley K. Yoder
• 金额: $ 2.79万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7918096
• 关键词: Address; Adult; Alleles; American; Animals; Antibodies; Architecture; Bed rest; Bone Density; Bone Development; Bone Matrix; Bone remodeling; Caenorhabditis elegans; Calcium; Calcium Channel; Calcium Signaling; Cations; Cells; Cilia; Communication; Complex; DEXA; Data; Defect; Deposition; Development; Dose; Dual-Energy X-Ray Absorptiometry; Ductal Epithelium; Epithelial Cells; Epithelial Physiology; Feasibility Studies; Functional disorder; Future; Gene Targeting; Genes; Gifts; Goals; Homeostasis; Image; Immunofluorescence Immunologic; Kidney; Lead; Link; Liver; Maintenance; Mechanical Stress; Mechanics; Mediating; Minerals; Molecular; Mus; Mutant Strains Mice; Mutation; Nephrology; Nitric Oxide; Organelles; Osteoblasts; Osteocalcin; Osteoclasts; Osteocytes; Osteogenesis; PKD2 protein; Pathology; Pathway interactions; Pattern; Phenotype; Physical activity; Physiological; Play; Polycystic Kidney Diseases; Positioning Attribute; Production; Regulation; Renal tubule structure; Reporter; Research; Research Activity; Rheumatism; Role; Sensory; Series; Signal Pathway; Signal Transduction; Skeleton; Societies; Space Flight; Spatial Distribution; Staging; Stress; Structure; System; Tamoxifen; Testing; Tissues; Transgenic Mice; Weight-Bearing state; abstracting; base; bone; bone cell; bone mass; congenic; fluid flow; fluorexon; forgetting; insight; mature animal; mutant; novel; null mutation; polycystic kidney disease 1 protein; programs; promoter; recombinase; release of sequestered calcium ion into cytoplasm; renal epithelium; response

Bone mass is sensitive to mechanical stresses such that under periods of reduced strain (i.e. spaceflight, bed rest, etc) bone density is reduced, while repetitive load bearing stresses (i.e. physical activity) on the skeleton lead to increased bone formation. The mechanism of this regulation is not clear, but is thought to involve mechanosensors that detect shear forces caused by fluid flow through the bone canaliculi in response to the compression of the bone. The osteocytes located in the bone matrix are thought to be the cells responsible for detecting this mechanical signal. The osteocyte has an elaborate communication system interconnecting with the osteoblasts that line the bone and mediate mineral deposition and with the osteoclasts that reabsorb bone. Thus, the osteocyte is ideally positioned to sense external stresses and elicit appropriate adaptive responses needed to remodel bone; however, the mechanism by which it mediates this sensory response is unknown. Based on data from ductal epithelium in the kidney, we predict that the mechanical stress placed on the Done will be perceived by a primary cilium located on the osteocyte. As in the kidney, the primary cilia would function as a mechanosensor detecting flow through deflection of the axoneme. We predict this will result in calcium entry through the cilium requiring the nonselective cation channel polycystin-2. As in the renal epithelium this would lead to the release of internal calcium stores. While the physiological consequence of the calcium signal in the kidney is unknown, in the case of bone we predict it will regulate pathways governing bone formation and reabsorption. It is these possibilities that we want to test in this Pilot and Feasibility study. Thus the goal of this application is to evaluate the importance of the primary cilium and polycystin-2 for normal bone homeostasis. We will first determine the position of the cilium on the osteocyte in relation to the canaliculi and evaluate whether the polycystin-2 channel is present on the cilia as seen in the tubules of the kidney. We will then disrupt cilia or polycystin-2 function in the osteocytes/osteoblast. Since congenic mutations that completely disrupt cilia formation or polycystin-2 function are not viable and die before bone development occurs, we will cross conditional floxed mutant alleles of Tg737 and Kif3a (cilia assembly genes) and polycystin-2 with transgenic mice expressing Cre recombinase from the osteocalcin (OC::Cre) promoter. We will also use an OC::Cre line where Cre activity will be tamoxifen inducible which will allow us to disrupt cilia/polycystin-2 function during development or in adults. The consequence of the loss of cilia and polycystin-2 function on bone development and maintenance will be assessed by microCT, DEXA, and by histomorphometry. Future research directions of this application would involve the isolation of osteocytes to evaluate their ability to respond to fluid flow in presence or absence of cilia and polycystin-2. In addition, we will assess the effect of cilia) dysfunction on pathways, such as IGF and NO, that are involved in the regulation of bone homeostasis. Overall, these analyses will provide novel insights into the mechanosensory role of the primary cilium on the osteocyte and how this cell regulates bone formation.

骨骼质量对机械应力敏感，以便在减少应变时（即太空飞行， 床休息等骨密度降低，而重复负载应力（即身体活动） 骨骼导致骨形成增加。该法规的机制尚不清楚，但被认为是 涉及机械传感器，该机械传感器检测到流经骨的流体引起的剪切力 对骨骼压缩的反应。骨基质中的骨细胞被认为是 负责检测该机械信号的细胞。骨细胞具有精致的交流 系统与骨头上的成骨细胞互连并介导矿物沉积，并与 破骨细胞可吸收骨骼。因此，骨细胞是理想的定位，可以感觉到外部压力和 引起重塑骨所需的适当自适应反应；但是，它的机制 介导这种感觉反应是未知的。 根据肾脏导管上皮的数据，我们预测机械应力放在 在骨细胞上的原发性纤毛会感知完成。与肾脏一样，主要的纤毛会 通过轴突偏转的机械传感器检测流动。我们预测这将导致 钙通过纤毛进入需要非选择性阳离子通道Polycystin-2。就像肾脏一样 上皮这将导致内部钙存储的释放。而生理后果 肾脏中的钙信号未知，就骨骼而言，我们预计将调节途径 治理骨形成和重吸收。我们想在此飞行员中测试的这些可能性 可行性研究。 因此，此应用的目的是评估主要纤毛和polycystin-2对 正常的骨体内平衡。我们将首先确定纤毛在骨细胞上的位置 canaliculi并评估在纤毛中是否存在多囊这汀 - 2通道 肾。然后，我们将破坏整骨细胞/成骨细胞中的纤毛或polycystin-2功能。自chanig 完全破坏纤毛形成或polycystin-2功能的突变是不可行的，在骨头之前死亡 发生发展，我们将跨有条件的TG737和KIF3A（Cilia组装 基因）和多囊这胞蛋白-2，带有从骨钙素表达CRE重组酶的转基因小鼠（OC :: CRE） 发起人。我们还将使用OC :: CRE系列，其中CRE活动将是他莫昔芬诱导的，这将使我们 在发育过程中或成年人中破坏纤毛/polycystin-2功能。纤毛损失的结果和 Polycystin-2在骨发育和维持方面的功能将通过Microct，Dexa以及通过 组织形态法。该应用的未来研究方向将涉及将骨细胞隔离为 评估他们在存在或不存在纤毛和多囊2的情况下对流体流动反应的能力。另外，我们 将评估纤毛功能障碍对IGF和NO等途径的影响 骨体内平衡的调节。总体而言，这些分析将为机械增强提供新的见解 主要纤毛在骨细胞上的作用以及该细胞如何调节骨形成。