DAP12 and ITAM-signals in Osteoclastogenesis

描述（由申请人提供）： 骨是一个动态的器官，通过造骨细胞和破骨细胞不断重塑，造新骨和破骨细胞降解骨。骨吸收是破骨细胞的独特功能，破骨细胞是一种来源于骨髓系造血细胞的细胞类型。破骨细胞的发育需要通过RANK（NF κ B活化受体）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）信号传导受体对共刺激信号的额外需求。有趣的是，我们还发现ITAM-适配器缺陷小鼠在由雌激素缺乏、低钙饮食或LPS（脂多糖）诱导的快速骨重建条件下丢失骨。因此，在这些骨重建的病理条件下，其他共刺激信号和/或受体可以替代破骨细胞生成中的ITAM-衔接子介导的信号。在破骨细胞生成中，认为ITAM信号受体主要提供Ca 2+信号，以在RANKL刺激期间激活破骨细胞生成的关键转录因子NFATc 1。我们有兴趣确定ITAM信号受体提供的其他信号，这些信号对破骨细胞生成也很重要，以帮助我们了解共刺激信号的要求。在其他细胞类型中，ITAM-信号也导致活性氧（ROS）的产生。活性氧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生成过程中刺激ROS产生。我们观察到，RANKL刺激的ROS产生在前破骨细胞不发生在ITAM衔接子的情况下。我们的建议将调查的假设，ITAM-适配器提供了一个Ca 2+信号和ROS信号的破骨细胞前体，共同需要破骨细胞的发生。我们认为，如果没有ITAM信号在破骨细胞前体，破骨细胞只能产生压力或炎症条件下，活性氧普遍。我们的假设表明，破骨细胞分化的基础和炎症条件下不同的刺激，并可能揭示破骨细胞的亚型，可以差异调节。我们提出以下具体目标：1。确定ROS刺激对ITAM信号通路的影响以及ROS增强破骨细胞生成的机制。2.确定RANKL刺激的破骨细胞生成过程中产生活性氧（ROS）对ITAM接头和ITAM接头信号的需求。3.确定在缺乏ITAM-接头信号的情况下快速破骨细胞生成过程中对ROS的需求。 公共卫生相关性： 破骨细胞的功能是终生吸收骨。包括骨质疏松症、类风湿性关节炎和癌症转移在内的许多疾病都涉及由于破骨细胞活化不当而导致的显著骨丢失。骨质疏松症的发病率随着年龄的增长而增加，在VA患者人群中很常见。骨质疏松性骨折的治疗是昂贵的，在骨质疏松性脊椎或髋部骨折的患者中，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压力或炎症条件下发育所需的信号与基础条件下破骨细胞发育所需的信号有何不同。我们的研究对于促进新疗法的开发以预防我们患者的早期骨丢失和骨折非常重要。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.