Mechanism of ITAM Signal Regulation in Osteoclasts

描述（由申请人提供）：通过成骨细胞的骨沉积和破骨细胞（OC）的骨吸收的仔细平衡来维持骨完整性。骨重建发生在整个生命和不平衡的形成和吸收导致人类疾病，包括骨质疏松症和牙周炎，一个使命的机构。我们正在研究破骨细胞中ITAM适配器信号的负调控。具体来说，我们正在研究磷酸酶可能调节ITAM-适配器，DAP 12，信号传导的可能性。我们发现了一种新的激活的DAP 12与含SH 2的肌醇-5 '-磷酸酶1（SHIP 1）的结合。我们的中心假设是SHIP 1在体外和体内OC发育和功能过程中调节DAP 12信号。具体目标1：确定SHIP 1抑制DAP 12信号传导的机制。这些研究旨在确定抑制DAP 12信号传导所需的SHIP 1功能结构域、近端激酶的作用以及SHIP 1负调控的特定下游信号传导途径。我们将确定SHIP 1在MCSF、RANKL和整合素刺激的下游负调节DAP 12中的作用。我们将研究DAP 12相关受体在介导SHIP 1-DAP 12相关性中的具体作用。具体目标2：确定SHIP 1对DAP 12信号传导的抑制如何影响OC在体外的再吸收、肌动蛋白环形成和存活功能。我们将确定在DAP 12刺激期间SHIP 1和DAP 12的细胞定位。具体目标3：确定SHIP 1对体内DAP 12信号传导的影响。我们将确定在用抗TREM 2抗体直接激活DAP 12或用TREM 2融合蛋白阻断DAP 12后，SHIP 1是否在体内调节DAP 12信号传导。具体目的4：确定SHIP 1和TREM 2/DAP 12在响应牙龈卟啉单胞菌（牙龈卟啉单胞菌）诱导的牙槽骨丢失中的作用。此外，我们将研究DAP 12和SHIP 1在体内慢性低剂量LPS治疗或牙龈卟啉单胞菌LPS急性刺激诱导的炎性小梁和牙槽骨丢失中的作用。这些研究不仅将阐明SHIP 1如何调节OC中的DAP 12信号传导，还将潜在地深入了解DAP 12如何在巨噬细胞和OC中作为激活和抑制信号发挥作用。这种理解可能会导致牙周病和骨质疏松症的新的治疗干预措施。项目叙述：这项研究的目的是确定一种抑制破骨细胞（溶解骨的细胞）活性所需的关键调节途径。这种理解可能会导致新的治疗干预慢性牙周病和骨质疏松症，与破骨细胞过度活动的疾病。

DESCRIPTION (provided by applicant): Bone integrity is maintained via the careful balance of bone deposition by osteoblasts and bone resorption by osteoclasts (OC). Bone remodeling occurs throughout life and imbalances in formation and resorption lead to human diseases including osteoporosis and periodontitis, a mission of the agency. We are studying the negative regulation of ITAM-adapter signals in osteoclasts. Specifically we are investigating the possibility that phosphatases may regulate ITAM-adapter, DAP12, signaling. We have found a novel association of activated DAP12 with SH2-containing inositol-5'-phosphatase 1 (SHIP1). Our central hypothesis is that SHIP1 regulates DAP12 signaling during OC development and function in vitro and in vivo. Specific Aim 1: Determine the mechanism by which SHIP1 inhibits DAP12 signaling. These studies aim to determine the functional domains of SHIP1 that are required for inhibition of DAP12 signaling, the role of proximal kinases, and the specific downstream signaling pathways negatively regulated by SHIP1. We will determine the role of SHIP1 in negatively regulating DAP12 downstream of MCSF, RANKL, and integrin stimulation. We will investigate the specific role of DAP12-associated receptors in mediating SHIP1- DAP12 association. Specific Aim 2: Determine how SHIP1 inhibition of DAP12 signaling affects the OC functions of resorption, actin ring formation and survival in vitro. We will determine the cellular localization of SHIP1 and DAP12 during DAP12 stimulation. Specific Aim 3: Determine the affect of SHIP1 on DAP12 signaling in vivo. We will determine whether SHIP1 regulates DAP12 signaling in vivo after direct activation of DAP12 with anti-TREM2 antibodies or blockade of DAP12 with TREM2-fusion protein. Specific Aim 4: Determine the role of SHIP1 and TREM2/DAP12 in response to Porphyromonas gingivalis (P. gingivalis) induced alveolar bone loss. Additionally we will investigate the roles of DAP12 and SHIP1 in inflammatory trabecular and alveolar bone loss induced by chronic low dose LPS treatment or acute stimulation with P. gingivalis LPS in vivo. These studies will not only elucidate how SHIP1 regulates DAP12 signaling in OC but will potentially give insights into how DAP12 can function as both an activating and inhibitory signal in macrophages and OC. This understanding might lead to novel therapeutic interventions in periodontal disease and osteoporosis. Project Narrative: The aim of proposed studies is to define a key regulatory pathway needed to inhibit activity of osteoclasts, cells that dissolve bone. This understanding might lead to novel therapeutic interventions in chronic periodontal disease and osteoporosis, diseases associated with excessive osteoclast activity.