Functional Analysis of NHA-oc/NHA2 in vivo

描述(申请人提供)：破骨细胞破坏骨骼对于正常的骨骼发育是必不可少的。破骨细胞缺乏会导致骨化症，其特征是骨量增加，可能会导致骨骼变形，严重时会导致死亡。另一方面，破骨细胞数量和活性的增加会导致骨吸收增加，并可能导致骨质疏松和其他溶骨性疾病。更好地了解破骨细胞形成、活性和存活的分子调控将为控制这些疾病的治疗干预提供新的靶点。最近，我们在体内和体外发现了一个在RANKL诱导破骨细胞分化过程中强烈上调的基因nha-oc/NHA2。NHA-oc/NHA2编码一种新的阳离子/质子反向转运蛋白(CPA)，它选择性地在破骨细胞中表达。NHA-oc/NHA2蛋白定位于线粒体，在那里它介导了钠依赖的线粒体肿胀。因此，nHA-oc/NHA2是第一个表征哺乳动物线粒体CPA2的基因。NHA-oc/NHA2的RNA沉默减少了破骨细胞的分化和骨吸收，有力地表明了NHA-oc/NHA2在这些过程中的作用。在目标1中，我们将通过在NHA-oc/NHA2基因座携带突变逆转录病毒插入的基因敲除小鼠模型来表征NHA-oc/NHA2在体内骨骼稳态中的作用。在目标2中，我们将研究NHA-oc/NHA2在体内病理性骨丢失中的作用。为此，我们将比较卵巢切除在野生型和纯合子突变小鼠中造成的骨丢失。NHA-oc/NHA2在体内对破骨细胞形成和骨吸收的影响将通过多种骨分析方法来确定。我们的目标是确定NHA-oc/NHA2在破骨细胞分化和功能中所起的作用，并最终在体内调节骨量。这些研究可能发现与人类骨化病发生发展相关的新的候选基因，并为抗吸收治疗提供一个重要的新靶点。 公共卫生相关性：破骨细胞是在正常的骨骼发育和维护过程中负责骨移除(‘吸收’)的细胞。相反，破骨细胞数量和/或活动异常会导致一系列疾病，从骨化症到骨质疏松症。该项目旨在确定我们在破骨细胞中发现的一种新基因NHA-oc/NHA2在体内破骨细胞形成、功能和最终调节骨量中的作用。这项工作将帮助我们设计基于干扰NHA-oc/NHA2活性的药物的适当的新疗法，以防止病理性骨丢失。

DESCRIPTION (provided by applicant): Bone destruction by osteoclasts is essential for normal bone development. Osteoclast deficiency leads to osteopetrosis, which is characterized by increased bone mass and may lead to bone deformities or in severe cases, to death. Increased numbers and activity of osteoclasts, on the other hand, cause increased bone resorption, and may lead to osteoporosis and other osteolytic diseases. A better understanding of the molecular regulation of osteoclast formation, activity, and survival will provide novel targets for therapeutic intervention in the control of these diseases. Recently, we identified a gene, nha-oc/NHA2, which is strongly up-regulated during RANKL-induced osteoclast differentiation in vitro and in vivo. nha-oc/NHA2 encodes a novel cation/proton antiporter (CPA) that is selectively expressed in osteoclasts. NHA-oc/NHA2 protein localizes to the mitochondria, where it mediates Na+dependent mitochondrial swelling. NHA-oc/NHA2 is therefore the first characterized mammalian mitochondrial CPA2. RNA silencing of nha-oc/nha2 reduces osteoclast differentiation and bone resorption, strongly indicating a role for NHA-oc/NHA2 in these processes. In Aim 1, we will characterize the role of NHA-oc/NHA2 in bone homeostasis in vivo by using a knockout mouse model carrying a mutagenic retroviral insertion in the nha-oc/NHA2 locus. In Aim 2, we will characterize the role of NHA-oc/NHA2 on pathological bone loss in vivo. For that, we will compare bone loss induced by ovariectomy in wild type and homozygous mutant mice. The effects of nha-oc/NHA2 on osteoclast formation and bone resorption in vivo will be determined using a variety of analytic approaches for bone. Our goal is to establish the role that NHA-oc/NHA2 plays in osteoclast differentiation and function, and ultimately in regulating bone mass in vivo. These studies may identify a new candidate gene involved in the development of human osteopetrosis as well as provide an important new target for anti-resorptive therapies. PUBLIC HEALTH RELEVANCE: Osteoclasts are cells that are responsible for bone removal ('resorption') during normal bone development and maintenance. In contrast, abnormal osteoclast numbers and/or activity cause a spectrum of diseases ranging from osteopetrosis to osteoporosis. This project seeks to determine the role of a novel gene that we have discovered in osteoclasts, termed 'NHA-oc/NHA2', in osteoclast formation, function and ultimately in regulating bone mass in vivo. This work will aid us in the design of appropriate new therapies based on drugs that interfere with NHA-oc/NHA2 activity for the prevention of pathological bone loss.