Signal Transduction Mechanism of Osteoclast Differentiation

描述(由申请人提供)： 骨形成和骨吸收的紧密平衡(称为骨重塑)维持骨骼的动态平衡。骨重建是成骨细胞和骨吸收破骨细胞共同作用的结果。这些功能相反的细胞通过旁分泌细胞信号网络进行交流。平衡的改变有利于多核破骨细胞的形成，导致骨破坏，如自身免疫性关节炎、牙周病、绝经后骨质疏松症、帕吉特氏病和骨肿瘤转移。另一方面，维持关键的骨稳态需要有秩序的成骨细胞和破骨细胞的供应。骨形态发生蛋白(BMPs)被用作促进骨再生的治疗措施。最近的报道表明，BMP通过成骨细胞或基质细胞间接作用于破骨细胞。骨形态发生蛋白-2(BMP-2)调节破骨细胞活性的确切机制是这一提议的关键问题。这将对设计治疗美国退伍军人常见的骨骼退行性疾病的药物具有治疗意义。BMP与其细胞表面受体相互作用激活Smad信号通路。我们已经证明了Smad信号在BMP-2诱导的成骨细胞表达集落刺激因子-1(CSF-1)中发挥了作用，CSF-1是破骨细胞形成的关键调节因子。我们的初步实验表明，Smad信号参与了BMP-2诱导的关键成骨细胞转录因子OSX的表达。此外，在这项研究中，我们首次证明了BMP-2增加了另一种关键的破骨细胞生成蛋白--核因子：B受体激活剂配体(RANKL)的表达，并激活了激活的T细胞核因子c1(NFATc1)，后者是破骨细胞生成的主要调节因子。本研究假设BMP-2通过激活转录因子OSX和NFATc1来调控集落刺激因子-1(CSF-1)和RANKL的表达，从而启动破骨细胞的吸收活动。在第一个特定目标中，我们将研究两个关键转录因子OSX和NFATc1在成骨细胞对BMP-2反应中调节破骨细胞基因(RANKL和CSF-1)表达的作用。在第二个特定目标中，我们将研究BMP-2诱导成骨细胞中OSX和NFATc1表达和激活的机制。在第三个特定目标中，我们将测试OSX和NFATc1在BMP-2诱导的破骨细胞形成中的功能参与。我们将使用免疫印迹、免疫组织化学、原位杂交、芯片分析和qRTPCR技术来检测BMP-2在激活这些导致破骨细胞分化的重要转录因子中的关键作用。在这项提案中，我们还将探索BMP-2诱导的microRNAs对OSX基因表达的新的转录后调控。我们的结果将展示BMP-2如何在成骨细胞中协调复杂的转录网络，以严格调控破骨细胞的激活。 公共卫生相关性： 拟议工作与退伍军人健康的相关性：在退伍军人和老年人群中，骨骼重塑动态平衡的破坏是骨相关疾病的重要决定因素。这些疾病中最常见的是骨质疏松症，它会导致频繁的骨折和极度的骨痛。超过200万美国男性患有破骨细胞活动异常导致的骨质疏松症。每年有8万男性髋部骨折，其中三分之一在一年内死亡。增加对破骨细胞形成机制及其活性的了解将导致合理的方法来管理骨丢失。这项建议的目的是为了了解破骨细胞活性的调节。这项建议中描述的实验结果将阐明特定转录因子的作用，这些转录因子调节成骨细胞介导的破骨细胞分化，并将有助于开发药物来调节许多骨疾病中的破骨细胞活性，这些疾病的主要病理是骨质破坏。

DESCRIPTION (provided by applicant): A tight balance of bone formation and bone resorption (termed as the bone remodeling) maintains the homeostasis of the skeleton. The bone remodeling occurs by concerted action of bone forming osteoblasts and bone resorbing osteoclasts. These functionally opposing cells communicate via a paracrine cell signaling network. A shift in the balance in favor of multi-nucleated osteoclast formation causes bone destruction as observed in diseases such as autoimmune arthritis, periodontosis, postmenopausal osteoporosis, Padget's disease and bone tumor metastasis. On the other hand, an orderly supply of osteoblasts and osteoclasts are needed for maintaining the critical bone homeostasis. The bone morphogenetic proteins (BMPs) are used as therapeutic measures for promoting bone regeneration. Recent reports suggest that BMPs act on osteoclasts indirectly through osteoblasts or stromal cells. The precise mechanism by which BMP type 2 (BMP-2) regulates the osteoclast activity is the key question in this proposal. This will have therapeutic implications in designing drugs for the bone degenerating diseases common to the war veterans in the USA. Interaction of BMP with its cell surface receptors activates Smad signaling pathway. We have demonstrated a role for Smad signaling in BMP-2-induced osteoblastic expression of the colony stimulating factor-1 (CSF-1), a key regulator of osteoclastogenesis. Our preliminary experiments demonstrate an involvement of Smad signaling in BMP-2-induced expression of the critical osteoblastic transcription factor, osterix (Osx). Furthermore, in this proposal, we show for the first time that BMP-2 increases osteoblastic expression of another critical osteoclastogenic protein, the receptor activator of NF:B ligand (RANKL) and activates nuclear factor of activated T cells c1 (NFATc1), a master regulator of osteoclastogenesis. The hypothesis to be tested in this proposal is that BMP-2 activates the transcription factors osterix (Osx) and NFATc1 to control the expression of colony stimulating factor-1 (CSF-1) and RANKL in order to initiate the resorptive activity by osteoclasts. In the first specific aim, we will investigate the role of two key transcription factors, Osx and NFATc1 in regulating expression of the osteoclastogenic genes (RANKL and CSF-1) in osteoblasts in response to BMP-2. In the second specific aim, we will examine the mechanism underlying BMP-2-induced expression and activation of Osx and NFATc1 in osteoblasts. In the third specific aim, we will test the functional involvement of Osx and NFATc1 in BMP-2-induced osteoclastogenesis. We will test critical involvement of BMP-2 in activating these important transcription factors leading to osteoclast differentiation using immunoblotting, immunohistochemistry, in situ hybridization, ChIP analysis and qRTPCR techniques. In this proposal we will also explore the novel post-transcriptional regulation of Osx gene expression by BMP-2- induced microRNAs. Our results will demonstrate how BMP-2 can orchestrate a complex transcriptional network in osteoblasts to tightly regulate osteoclast activation. PUBLIC HEALTH RELEVANCE: Relevance of the proposed work to Veteran's Health: The disruption of bone remodeling homeostasis is an important determinant of bone related diseases in the veterans and the elderly populations. The most prevalent of these diseases is osteoporosis that gives rise to frequent bone fractures and extreme bone pain. More than 2 million American men suffer from osteoporosis brought about by abnormal osteoclast activity. 80,000 men have hip fractures every year of which one-third dies within a year. Increased understanding of the mechanisms involved in osteoclast formation and their activity will lead to rational approaches to manage bone loss. The objectives of this proposal are aimed at understanding the regulation of osteoclast activity. The results obtained from the experiments described in this proposal will elucidate the role of specific transcription factors, which regulate the osteoblast-mediated osteoclast differentiation and will help in developing drugs to regulate osteoclast activity in many bone diseases where destruction of bone is the main pathology.