Bone matrix and bone resorption

描述(由申请人提供)：此申请是对NOT-OD-09-058的响应，“NIH宣布恢复法资金可用于竞争性修订申请。”这是对RO1 DE 007444“骨基质和骨吸收”的竞争性修订申请，在该申请下，我们正在研究我们发现的一种名为plekhm1的蛋白质的破骨细胞生物学。有证据表明，plekhm1是破骨细胞正常运输囊泡、分泌和骨吸收所必需的。在大鼠模型和人类患者中，截短plekhm1蛋白的突变会导致骨化病和破骨细胞不活跃。相比之下，我们的合作者最近在一名人类患者身上发现了一个点突变R714C，它似乎会导致破骨细胞过度活动。这会导致破骨细胞和骨质疏松骨骼的分泌活动增加。目前的项目正在研究与plekhm1相互作用的结合伙伴，以控制破骨细胞中的囊泡运动和分泌，以及生物活性脂质对其的调节。这项提议将为我们正在进行的项目增加一个新的具体目标。我们假设plekhm1-R714C突变是一种显性的功能获得突变，会导致过度的骨丢失。我们将在新的特定目标下通过建立一个小鼠模型来检验这一假设，在该模型中，内源性的野生型plekhm1被带有plekhm1-R714C的基因敲入所取代。小鼠的骨骼表型将通过DEXA、pQCT和microCT进行评估。还将测量血清TRAP水平，以确定小鼠品系在多大程度上复制了点突变杂合子的人类患者。透射电子显微镜和免疫电子显微镜研究将检测突变的破骨细胞。来自基因敲除小鼠的细胞将被用来研究破骨细胞生物学和plkhm1结合的变化，以及由点突变引起的调控。将聘请一名新的博士后来实施这一项目，为期2年。我们还申请资金购买荧光显微镜，使用它我们可以对细胞培养中标记蛋白的表达进行常规筛选，并进行去卷积共聚焦成像实验，以筛选候选蛋白与plekhm1共定位，作为破骨细胞正常的囊泡运输、分泌和骨吸收所需的复合体的一部分。 公共卫生相关性：项目简介：广泛性骨骼疾病，包括骨质疏松症、关节炎、牙周骨病、关节置换松动和肿瘤转移到骨，都有共同的骨丢失超过骨形成。骨丢失是由称为破骨细胞的细胞进行的，我们对破骨细胞如何工作了解得越深入，潜在的治疗方法就会出现更多的机会来控制它们的活动。这项提议旨在研究我们在动物模型和人类患者中发现的导致骨病的一种新基因。更深入地了解这种基因及其如何控制破骨细胞的骨吸收活动将使人们从总体上更好地了解骨生物学，并可能导致治疗这些非常常见的骨病的新方法。

DESCRIPTION (provided by applicant): This application is in response to NOT-OD-09-058, "NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications." It is a competitive revision request for grant RO1 DE 007444, "Bone matrix and bone resorption," under which we are investigating the osteoclast cell biology of a protein we discovered called plekhm1. Evidence indicates that plekhm1 is required for normal trafficking of vesicles, secretion, and bone resorption by osteoclasts. Mutations that truncate the plekhm1 protein cause osteopetrosis and inactive osteoclasts in a rat model and in human patients. In contrast, a point mutation, R714C, was recently discovered by our collaborators in a human patient, which appears to cause excessive osteoclast activity. This leads to increased secretory activity by osteoclasts and an osteopenic skeleton. The current project is examining binding partners that interact with plekhm1 to control vesicle movement and secretion in osteoclasts, as well as its regulation by bioactive lipids. This proposal would add a new specific aim to our ongoing project. We hypothesize that the plekhm1-R714C mutation is a dominant, gain-of-function mutation that leads to excessive bone loss. We will test this hypothesis under the new specific aim by generating a mouse model in which the endogenous, wild type plekhm1 is replaced by gene knock-in with plekhm1-R714C. The skeletal phenotype of the mice will be evaluated by DEXA, pQCT and microCT. Serum TRAP levels will also be measured to determine the extent to which the mouse line phenocopies the human patient who is heterozygous for the point mutation. TEM and immune-EM studies will examine mutant osteoclasts. Cells derived from the knock-in mice will be used to investigate the alterations in osteoclast biology and plekhm1 binding and regulation caused by the point mutation. A new post-doctoral fellow will be hired to carry out this project, which will last for 2 years. We are also requesting funds to purchase a fluorescence microscope with which we can both perform routine screening for tagged protein expression in cell cultures and perform deconvolution confocal imaging experiments to screen candidate proteins for co-localization with plekhm1, as part of a complex required for normal vesicle trafficking, secretion, and bone resorption by osteoclasts. PUBLIC HEALTH RELEVANCE: Project narrative: Widespread bone disorders, including osteoporosis, arthritis, periodontal bone disease, joint replacement loosening, and tumor metastasis to bone, all have in common bone loss that exceeds bone formation. Bone loss is carried out by cells called osteoclasts, and the more deeply we understand how osteoclasts work, the more opportunities will arise for potential therapeutics to control their activity. This proposal aims to investigate a new gene we discovered which causes bone disease in animal models and in human patients. Deeper understanding of this gene and how it controls the bone resorbing activity of osteoclasts will give a better knowledge of bone biology in general, and potentially will lead to new ways to treat these all-too-common bone disorders.