Regulatory Mechanisms of Implant-Induced Osteolysis

描述（由申请人提供）：由于衰老和/或病理状况如关节炎和骨质疏松症导致的大关节退行性疾病导致手术干预方法主要是全关节置换术（TJR）。然而，随着时间的推移，种植体产生的磨损碎片会引起炎症反应，最终导致骨溶解和种植体失败。失败的关节植入物的后续翻修手术通常更困难，并且与发病率和死亡率增加有关，特别是对于骨骼较弱的老年患者。因此，近年来对诊断、预防和/或治疗TJR并发症的有效方法的需求日益增加。因此，更好地了解导致关节衰竭的病理和溶骨事件的过程和机制对于提供适当的预防和治疗对策至关重要。种植体磨损碎片的病理反应是这一现象的主要组成部分，目前正在深入研究中。包括我们在内的几个小组最近的工作已经确定了促进炎症性骨溶解的重要细胞实体和分泌因子。在以前的工作中，我们已经表明PMMA颗粒通过刺激破骨细胞前体的主要途径，主要是NF-？B和MAP激酶。前一途径需要组装包括IKK1、IKK2和IKK？，也被称为NEMO。我们最近已经证明干扰NF-？B和MAPK激活途径通过引入抑制剂和诱饵分子，在实验性颅骨骨溶解和炎症性关节炎小鼠模型中阻碍pmma诱导的骨溶解。在我们最近的工作中，我们发现PMMA颗粒直接激活上游转化生长因子β活化激酶-1 (TAK1)， TAK1是信号转导级联反应的关键调节剂，导致NF-？B和AP-1因子。更重要的是，我们发现PMMA颗粒诱导TAK1与NEMO、RIP1和UBC13结合。此外，我们发现PMMA颗粒诱导TRAF6与NEMO结合，缺乏TRAF6会显著减弱NEMO的泛素化。我们进一步证明PMMA诱导NF-？B和MAPK在tak1缺失和NEMO突变细胞中受损。这些反应不受TNF或RANKL的辅助。总之，这些结果使我们假设PMMA颗粒可能诱导了NEMO、RIP1和其他靶蛋白的k63连锁泛素化，这些事件可能由TRAF6、TAK1和UBC13介导。与这一假设相关的是，已有文献表明，LPS、IL-1和RANKL信号传导的关键介质TRAF6是泛素连接酶。在单独的研究中，进一步确定了多种上游信号增强了以TAK1/TABs/RIP/NEMO/UBC复合物为主的泛素化信号网络。因此，我们建议研究以下具体目标：1。描述pmma诱导的NEMO调控的分子步骤。2. 研究TRAF6/TAK1/NEMO多聚泛素化事件是否介导pmma诱导的破骨细胞发生。3. 确定抑制POLY-UB-NEMO信号传导对pmma诱导的颅骨骨溶解的影响。

DESCRIPTION (provided by applicant): Degenerative conditions of large weight bearing joints resulting from aging and/or pathologic conditions such as arthritis and osteoporosis lead to surgical intervention approaches chiefly total joint replacement (TJR). However, implant-derived wear debris occurs with time causing inflammatory responses culminating with osteolysis and failure of implants. Subsequent revision surgery of the failing joint implant, is often more difficult, and associated with increased morbidity and mortality especially for aging patients with weaker bones. Therefore, the need for effective approaches to diagnose, prevent, and/or treat complications of TJR have risen in recent years. Thus, better understanding of the processes and mechanisms underlying pathologic and osteolytic events leading to joint failure is essential to provide appropriate preventive and therapeutic countermeasures. The pathologic response to implant wear-debris constitutes a major component of this phenomenon and is under intense investigation. Recent work by several groups including ours has identified important cellular entities and secreted factors that contribute to inflammatory osteolysis. In previous work, we have shown that PMMA particles contribute to inflammatory osteolysis through stimulation of major pathways in osteoclast precursors, primarily NF-?B and MAP kinases. The former pathway requires assembly of large IKK complex encompassing IKK1, IKK2, and IKK?, also known as NEMO. We have shown recently that interfering with the NF-?B and MAPK activation pathways, through introduction of inhibitors and decoy molecules, impede PMMA-induced osteolysis in mouse models of experimental calvarial osteolysis and inflammatory arthritis. In our recent work, we found that PMMA particles directly activate the upstream transforming growth factor beta activated kinase-1 (TAK1) which is a key regulator of signal transduction cascades leading to activation of NF-?B and AP-1 factors. More importantly, we found that PMMA particles induce TAK1 binding to NEMO, RIP1, and UBC13. In addition, we show that PMMA particles induced TRAF6 binding to NEMO and lack of TRAF6 significantly attenuates NEMO ubiquitination. We further demonstrate that PMMA induction of NF-?B and MAPK is impaired in TAK1-null and NEMO mutant cells. These responses were not aided by TNF or RANKL. Altogether, these results led us to hypothesize that PMMA particles maybe inducing K63-linked ubiquitination of NEMO, RIP1, and other target proteins, events likely mediated by TRAF6, TAK1 and UBC13. Relevant to this hypothesis, it has been documented that a key mediator of LPS, IL-1, and RANKL signaling, namely TRAF6, is ubiquitin ligase. In separate studies, it was further established that a variety of upstream signals augment ubiquitination-based signaling network dominated by TAK1/TABs/RIP/NEMO/UBC complex. Thus, we propose to investigate the following specific aims: 1. Delineate the molecular steps underlying PMMA-induced regulation of NEMO. 2. Investigate if TRAF6/TAK1/NEMO POLYUBIQUITINATION events mediate PMMA-induced osteoclastogenesis. 3. Determine the effect of inhibiting POLY-UB-NEMO signaling on PMMA-induced calvarial osteolysis. PUBLIC HEALTH RELEVANCE: Total joint implant failure is attributed, at least in part, to orthopedic particle-induced osteolysis. The mechanisms underlying this pathologic condition remain unclear. Our recent work has implicated heightened osteoclast activity, owing to enhanced intracellular activation of NF-?B and MAP kinase pathways, as the leading cause for inflammatory osteolysis. We provide evidence that key mediators of these two pathways, including TGF2-activating kinase (TAK1), IKK?/NEMO, E2-ligases such as Ubc13, and key ubiquitin-dependent events are induced by polymethylmethacrylate (PMMA) particles in osteoclast precursors. Thus, we will utilize in vitro and in-vivo approaches including genetically-modified mice to examine the role of ubiquitin-mediated events that contribute to exacerbation of PMMA-induced inflammatory osteolysis. Our proposal addresses novel questions and holds promise to identify novel selective anti-osteolytic therapies.