Regulatory Mechanisms of Implant-Induced Osteolysis

描述（由申请人提供）：由于老化和/或病理状况（如关节炎和骨质疏松症）导致的大型承重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，植入物产生的磨损碎屑会引起炎症反应，最终导致骨溶解和植入物失效。失败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相关，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有所增加。因此，更好地了解导致关节衰竭的病理和溶骨性事件的过程和机制，对于提供适当的预防和治疗对策至关重要。对种植体磨损碎片的病理反应构成了这种现象的主要组成部分，正在进行深入研究。包括我们在内的几个小组最近的工作已经确定了导致炎性骨质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颗粒有助于通过刺激破骨细胞前体，主要是NF-？B和MAP激酶。前一种途径需要组装包含IKK 1、IKK 2和IKK？的大型IKK复合物，也被称为NEMO。我们最近已经表明，干扰NF-？在实验性颅骨溶解和炎症性关节炎的小鼠模型中，通过引入抑制剂和诱饵分子，B和MAPK激活途径阻止了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激活激酶-1（TAK 1），这是导致NF-κ B激活的信号转导级联的关键调节因子。B和AP-1因子。更重要的是，我们发现PMMA颗粒诱导TAK 1与NEMO、RIP 1和UBC 13结合。此外，我们发现PMMA颗粒诱导TRAF 6与NEMO结合，并且TRAF 6的缺乏显著减弱NEMO泛素化。我们进一步证明，PMMA诱导NF-？B和MAPK在TAK 1缺失和NEMO突变细胞中受损。TNF或RANKL对这些反应无辅助作用。总之，这些结果使我们假设PMMA颗粒可能诱导NEMO、RIP 1和其他靶蛋白的K63连接的泛素化，这些事件可能由TRAF 6、TAK 1和UBC 13介导。与该假设相关，已经记录了LPS、IL-1和RANKL信号传导的关键介质，即TRAF 6，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由TAK 1/TABs/RIP/NEMO/UBC复合物主导的基于泛素化的信号网络。因此，我们建议调查以下具体目标：1。描述PMMA诱导NEMO调控的分子步骤。2.研究TRAF 6/TAK 1/NEMO多泛素化事件是否介导PMMA诱导的破骨细胞生成。3.确定抑制POLY-UB-NEMO信号传导对PMMA诱导的颅骨骨质溶解的影响。 公共卫生相关性：全关节植入物失效至少部分归因于骨科颗粒诱导的骨质溶解。这种病理状况的机制尚不清楚。我们最近的工作有牵连的破骨细胞活性的提高，由于增强细胞内激活NF-？B和MAP激酶途径，是炎性骨质溶解的主要原因。我们提供的证据表明，这两个途径的关键介质，包括TGF 2激活激酶（TAK 1），IKK？/ NEMO，E2-连接酶如Ubc 13，和关键的泛素依赖性事件是由聚甲基丙烯酸甲酯（PMMA）颗粒在破骨细胞前体中诱导的。因此，我们将利用体外和体内的方法，包括基因修饰的小鼠，以检查的作用，泛素介导的事件，有助于恶化PMMA诱导的炎性骨质溶解。我们的提案解决了新的问题，并有望确定新的选择性抗溶骨性疗法。

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.