Regulatory Mechanisms of Implant-Induced Osteolysis

描述(申请人提供)：由于老化和/或关节炎和骨质疏松症等病理条件导致的大承重关节的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屑会引起炎症反应，最终导致骨溶解和种植体失败。随后对失败的关节植入物进行翻修手术往往更加困难，并伴随着发病率和死亡率的增加，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事件的过程和机制对于提供适当的预防和治疗对策是至关重要的。对种植体磨屑的病理反应构成了这一现象的主要组成部分，目前正在进行深入的研究。包括我们在内的几个小组最近的工作已经确定了重要的细胞实体和有助于炎性骨溶解的分泌因子。在以前的工作中，我们已经证明PMMA颗粒通过刺激破骨细胞前体的主要途径，主要是核因子-β和MAP激酶，促进炎性骨溶解。前者需要组装大的IKK复合体，包括IKK1、IKK2和IKK？，也称为NEMO。我们最近在实验性颅骨溶解和炎性关节炎小鼠模型中发现，通过引入抑制剂和诱骗分子来干扰NF-B和MAPK的激活途径，可以阻止PMMA诱导的骨溶解。在我们最近的工作中，我们发现PMMA颗粒直接激活上游的转化生长因子β激活激酶-1(TAK1)，它是导致核因子-β和AP-1因子激活的信号转导级联的关键调节因子。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显著减弱NEMO泛素化。我们进一步证明，在TAK1缺失和Nemo突变细胞中，PMMA对NF-？B和MAPK的诱导是受损的。这些反应不受肿瘤坏死因子或RANKL的帮助。综上所述，这些结果导致我们假设PMMA颗粒可能诱导K63连接的NEMO、RIP1和其他靶蛋白的泛素化，这一事件可能由TRAF6、TAK1和UBC13介导。与这一假说相关的是，泛素连接酶是内毒素、IL-1和RANKL信号转导的关键介质，即TRAF6。在单独的研究中，进一步证实了各种上游信号增强了以泛素化为基础的信号网络，该网络由TAK1/Tabs/RIP/Nemo/UBC复合体主导。因此，我们建议研究以下具体目标：1.描述PMMA诱导NEMO调控的分子步骤。2.探讨TRAF6/TAK1/NEMO多聚体是否参与PMMA诱导的破骨细胞分化。3.确定抑制Poly-UB-NEMO信号通路对PMMA诱导的颅骨溶解的影响。 公共卫生相关性：关节植入物的全部失败至少部分归因于骨科颗粒诱导的骨溶解。这种病理状态背后的机制尚不清楚。我们最近的工作表明，破骨细胞的活性增加，这是由于核因子-β和MAP激酶通路在细胞内的激活增强，是炎症性骨溶解的主要原因。我们提供的证据表明，破骨细胞前体中的聚甲基丙烯酸甲酯(PMMA)颗粒诱导了这两个途径的关键介质，包括TGF2激活激酶(TAK1)、IKK？/NEMO、E2连接酶(如UBC13)和关键的泛素依赖事件。因此，我们将利用包括转基因小鼠在内的体外和体内方法来研究泛素介导的事件在加剧PMMA诱导的炎性骨溶解中的作用。我们的建议解决了新的问题，并有望找到新的选择性抗骨溶解疗法。

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.