DAP12 and ITAM-signals in Osteoclastogenesis

描述(由申请人提供)： 项目概述骨是一个动态的器官，由形成新骨的成骨细胞和降解骨骼的破骨细胞不断重塑。骨的吸收是破骨细胞的独特功能，破骨细胞是一种来源于髓系造血细胞的细胞类型。破骨细胞的发育需要通过RANK(激活NFkappaB的受体)和c-FMS(巨噬细胞集落刺激因子受体，M-CSF)的受体刺激。最近，我们和其他人证明了在正常的破骨细胞分化和功能过程中，通过ITAM(基于免疫受体酪氨酸的激活基序)信号受体对共刺激信号的额外要求。有趣的是，我们还发现，在雌激素缺乏、低钙饮食或脂多糖(LPS)诱导的快速骨重建条件下，ITAM适配器缺陷小鼠会丢失骨骼。因此，在这些骨重建的病理条件下，其他共刺激信号和/或受体可以替代ITAM适配器介导的信号在破骨细胞形成中的作用。在破骨细胞形成中，ITAM信号受体被认为主要是在RANKL刺激过程中提供钙信号来激活破骨细胞发生的关键转录因子NFATc1。我们感兴趣的是确定ITAM还提供了哪些其他信号-信号受体，这些信号受体对破骨细胞的形成也是至关重要的，以帮助我们了解对共刺激信号的要求。在其他类型的细胞中，ITAM-信号也导致活性氧物种(ROS)的产生。在体内和体外，ROS已被证明是破骨细胞分化和功能的重要调节因子。RANKL已被证明在破骨细胞形成过程中刺激ROS的产生。我们观察到，在没有ITAM适配器的情况下，RANKL刺激破骨前细胞产生ROS不会发生。我们的建议将研究这样一种假设，即ITAM适配器为破骨细胞前体提供钙信号和ROS信号，这两种信号一起是破骨细胞发生所必需的。我们认为，在破骨细胞前体中没有ITAM信号，破骨细胞只能在应激或炎症条件下产生，在这些条件下，活性氧普遍存在。我们的假设表明，破骨细胞在基础和炎症条件下在不同的刺激下分化，并可能揭示出可以差异调节的破骨细胞亚型。我们提出了以下具体目标：1.确定ROS刺激对ITAM信号通路的影响以及ROS促进破骨细胞生成的机制。2.确定在RANKL刺激的破骨细胞形成过程中产生活性氧(ROS)所需的ITAM-接头和ITAM接头信号。3.在没有ITAM-Adapter信号的情况下，确定快速破骨细胞形成过程中对ROS的需求。 公共卫生相关性： 项目叙事破骨细胞终生发挥吸收骨的功能。许多疾病，包括骨质疏松症、类风湿性关节炎和癌症转移，都涉及到由于破骨细胞激活不当而导致的大量骨丢失。骨质疏松症的发病率在男性和女性中随着年龄的增长而增加，在VA患者群体中很常见。骨质疏松性骨折的治疗费用昂贵，骨质疏松性椎体或髋部骨折患者的发病率和死亡率大大增加。我们感兴趣的是确定破骨细胞在应激或炎症条件下发展所需的信号与在基础条件下发展破骨细胞的信号有何不同。我们的研究对于促进新疗法的发展以预防患者早期骨丢失和骨折具有重要意义。我们的建议直接适用于指定的优先研究领域，包括老龄化、慢性病、癌症和退行性关节疾病。

DESCRIPTION (provided by applicant): Project Summary Bone is a dynamic organ, undergoing constant remodeling by osteoblasts that make new bone, and osteoclasts that degrade bone. The resorption of bone is the unique function of the osteoclast, a cell type derived from hematopoietic cells in the myeloid lineage. Osteoclast development requires receptor stimulation through RANK (receptor for activation of NFkappaB), and c-fms (receptor for macrophage colony stimulating factor, M-CSF). Recently, we and others demonstrated an additional requirement for costimulatory signals through ITAM (immunoreceptor tyrosine based activation motif) signaling receptors during normal osteoclast differentiation and function. Interestingly we also found that ITAM-adapter deficient mice lose bone under conditions of rapid bone remodeling induced by estrogen deficiency, a low calcium diet or LPS (lipopolysaccharide). Thus, other costimulatory signals and/or receptors can substitute for ITAM-adapter mediated signals in osteoclastogenesis under these pathologic conditions of bone remodeling. In osteoclastogenesis, ITAM-signaling receptors have been thought to primarily provide a Ca2+ signal to activate the critical transcription factor for osteoclastogenesis, NFATc1 during RANKL stimulation. We are interested in determining what other signals are provided by ITAM-signaling receptors that are also critical for osteoclastogenesis to help us understand the requirements for costimulatory signals. In other cell types, ITAM- signals also lead to production of reactive oxygen species (ROS). ROS have been previously demonstrated to be important regulators of osteoclast differentiation and function in vitro and in vivo. RANKL has been shown to stimulate ROS production during osteoclastogenesis. We observed that RANKL stimulated ROS production in preosteoclasts does not occur in the absence of ITAM adapters. Our proposal will investigate the hypothesis that ITAM-adapters provide both a Ca2+ signal and a ROS signal to osteoclast precursors that together are required for osteoclastogenesis. We propose that without ITAM- signaling in osteoclast precursors, osteoclasts can only be generated under stressful or inflammatory conditions where reactive oxygen species are prevalent. Our hypothesis suggests that osteoclasts differentiate under distinct stimuli under basal and inflammatory conditions, and may reveal subtypes of osteoclasts that can be differentially regulated. We propose the following specific aims: 1. Determine the effect of ROS stimulation on the ITAM-signaling pathway and the mechanism of enhanced osteoclastogenesis by ROS. 2. Determine the requirement for ITAM-adapters and ITAM adapter signals for production of reactive oxygen species (ROS) during RANKL stimulated osteoclastogenesis. 3. Determine the requirement for ROS during rapid osteoclastogenesis in the absence of ITAM- adapter signals. PUBLIC HEALTH RELEVANCE: Project Narrative Osteoclasts function to resorb bone throughout life. Many diseases including osteoporosis, rheumatoid arthritis, and cancer metastases involve significant bone loss due to inappropriate osteoclast activation. The incidence of osteoporosis increases with aging in men and women and is common in the VA patient population. Treatment of osteoporotic fractures is costly with greatly increased morbidity and mortality in patients with an osteoporotic vertebral or hip fracture. We are interested in determining how the signals required for osteoclasts to develop under stressful or inflammatory conditions differ from the signals that develop osteoclasts under basal conditions. Our studies are important to facilitate development of novel therapeutics to prevent early bone loss and fracture in our patients. Our proposal directly applies to the designated priority areas of research including aging, chronic disease, cancer and degenerative joint disease.