Signal Transduction Mechanism of Osteoclast Differentiation

描述（由申请人提供）： 骨形成和骨吸收（称为骨重建）的紧密平衡维持骨骼的稳态。骨重建是由成骨细胞和破骨细胞共同作用的结果。这些功能相对的细胞通过旁分泌细胞信号网络进行通信。有利于多核破骨细胞形成的平衡的转变导致骨破坏，如在诸如自身免疫性关节炎、牙周病、绝经后骨质疏松症、Padget病和骨肿瘤转移的疾病中观察到的。另一方面，需要成骨细胞和破骨细胞的有序供应来维持关键的骨稳态。骨形态发生蛋白（BMP）被用作促进骨再生的治疗措施。最近的研究表明，骨形成蛋白通过成骨细胞或基质细胞间接作用于破骨细胞。BMP-2调节破骨细胞活性的确切机制是本研究的关键问题。这将对设计治疗美国退伍军人常见的骨退化疾病的药物具有治疗意义。 BMP与其细胞表面受体的相互作用激活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破骨细胞生成的关键调节因子殖民地刺激因子1（CSF-1）中的作用。我们的初步实验表明，Smad信号参与BMP-2诱导的关键成骨细胞转录因子osterix（Osx）的表达。此外，在该提案中，我们首次表明，BMP-2增加成骨细胞表达的另一个关键的破骨细胞生成蛋白，NF受体激活剂：B配体（RANKL）和活化的T细胞c1（NFATc 1），一个主调节破骨细胞生成的核因子。在该提议中待检验的假设是BMP-2激活转录因子osterix（Osx）和NFATc 1以控制集落刺激因子-1（CSF-1）和RANKL的表达，从而启动破骨细胞的吸收活性。在第一个具体的目标，我们将调查的两个关键的转录因子，Osx和NFATc 1在调节破骨细胞基因（RANKL和CSF-1）在成骨细胞中的BMP-2的表达的作用。在第二个具体目标中，我们将研究BMP-2诱导成骨细胞中Osx和NFATc 1表达和激活的机制。在第三个具体目标中，我们将测试Osx和NFATc 1在BMP-2诱导的破骨细胞生成中的功能参与。我们将使用免疫印迹、免疫组织化学、原位杂交、ChIP分析和qRTPCR技术检测BMP-2在激活这些重要转录因子导致破骨细胞分化中的关键参与。在这个提议中，我们还将探索BMP-2诱导的microRNA对Osx基因表达的新的转录后调节。我们的研究结果将证明BMP-2如何在成骨细胞中协调复杂的转录网络，以严格调节破骨细胞的激活。

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.