Mechanism of ITAM Signal Regulation in Osteoclasts

描述(申请人提供)：通过小心平衡成骨细胞的骨沉积和破骨细胞(OC)的骨吸收来维持骨的完整性。骨重建发生在一生中，形成和吸收的不平衡会导致人类疾病，包括骨质疏松症和牙周炎，这是该机构的使命。我们正在研究破骨细胞中ITAM-适配器信号的负调控。具体地说，我们正在研究磷酸酶可能调节ITAM适配器DAP12信号的可能性。我们发现了激活的DAP12与SH2-含肌醇-5‘-磷酸酶1(SHIP1)的新关联。我们的中心假设是SHIP1在体外和体内的OC发育和功能过程中调节DAP12信号。具体目标1：确定SHIP1抑制DAP12信号转导的机制。这些研究旨在确定SHIP1抑制DAP12信号所需的功能结构域、近端激酶的作用以及SHIP1负调控的特定下游信号通路。我们将确定SHIP1在负调控MCSF、RANKL和整合素刺激下游的DAP12中的作用。我们将研究DAP12相关受体在介导SHIP1-DAP12关联中的特定作用。特定目的2：确定SHIP1抑制DAP12信号通路如何影响OC的吸收、肌动蛋白环形成和体外存活功能。我们将确定SHIP1和DAP12在DAP12刺激过程中的细胞定位。具体目的3：确定SHIP1在体内对DAP12信号转导的影响。我们将在用抗TREM2抗体直接激活DAP12或用TREM2融合蛋白阻断DAP12之后，确定SHIP1是否调节体内的DAP12信号转导。特异性目的4：探讨SHIP1和TREM2/DAP12在牙龈卟啉单胞菌(P.gigivalis)诱导的牙槽骨丢失中的作用。此外，我们还将在体内研究DAP12和SHIP1在慢性小剂量脂多糖治疗或牙龈假单胞菌急性刺激所致的炎性小梁和牙槽骨丢失中的作用。这些研究不仅将阐明SHIP1如何调节OC中的DAP12信号，而且可能为DAP12如何在巨噬细胞和OC中同时发挥激活和抑制信号的作用提供深入的见解。这一认识可能导致牙周病和骨质疏松症的新的治疗干预措施。项目简介：建议研究的目的是确定抑制破骨细胞活性所需的关键调控途径，破骨细胞是溶解骨骼的细胞。这一认识可能导致对慢性牙周病和骨质疏松症的新的治疗干预措施，这些疾病与过度的破骨细胞活动有关。

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.