Mechanisms of Physiologic and Pathologic Osteoclastogenesis

破骨细胞发生的生理和病理机制

基本信息

批准号：
9889901
负责人：
YOUSEF ABU-AMER
金额：
$ 33.55万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-04-01 至 2023-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9889901
关键词：
Address Alanine Autophagocytosis Binding Biochemical Bone Marrow Cell physiology Cells Complex Cues Disease Elements Exposure to Feedback Genes Homeostasis IL6 gene Impairment In Vitro Inflammation Inflammation Mediators Inflammatory Interferons Joints Knock-in Knock-in Mouse Knowledge Lead Lysine Mediating Modality Modeling Modification Molecular Molecular Profiling Molecular Target Mus Mutate Mutation Mutation Analysis Myelogenous NF-kappa B Nuclear Osteoclasts Osteolysis Osteopenia Outcome Pathologic Pathology Pathway interactions Phosphotransferases Physiological Polyubiquitination Post-Translational Protein Processing Process Production Proteins Proteomics Regulation Research Role Side Signal Pathway Signal Transduction Signaling Molecule Signaling Protein Site Skeletal Development Specificity Stimulus System TNF gene Ubiquitin Like Proteins base bone bone loss cell type combat cytokine experimental study gain of function in vivo joint destruction long bone macrophage monocyte mutant novel novel therapeutics osteoclastogenesis progenitor protein complex recruit response scaffold skeletal subchondral bone targeted treatment transcription factor

项目摘要

ABSTRACT: The transcription factor NF-kB is expressed ubiquitously in all cell types and is readily activated by numerous factors and cytokines. Baseline NF-kB activity is essential for skeletal development and physiologic cellular functions. In contrast, its exacerbated and often uncontrolled activity during inflammation leads to undesired harmful effects with major dysfunctional consequences including osteolysis. Hence, therapies targeting NF-kB have been highly pursued to combat most inflammatory diseases. Unfortunately, most available therapies are inefficient owing to lack of selectivity in such complex and ubiquitous signaling pathway wherein the essential beneficial functions of NF-kB are blocked along side the harmful effects leading to detrimental outcomes. Therefore, there is an unmet need to decode NF-kB signaling to identify specific targets that assign signal specificity and distinguish between physiologic and pathologic functions. To address this critical knowledge gap, we focused on RANKL-induced osteoclastogenesis as a proof of concept and set out to decipher the NF-kB molecular machinery and identify the signal-specific molecular signature that controls this response in osteoclast progenitors and maintains skeletal homeostasis. We hypothesize that the IKK scaffold IKKγ/NEMO serves as a platform that site-specifically assembles unique signal activating or suppressing protein complexes in cell and stimulus specific manners. This hypothesis is based on recent advances implicating NEMO as a scaffold that integrates signaling molecules in response to a wide range of stimuli at lysine (K) specific sites (refer to Fig 2). These modifications include, lysine poly-ubiquitination, SUMOylation, and according to our novel finding, ISGylation; a process of attaching the ubiquitin-like protein, ISG15 (IFN-stimulated gene) to target proteins. We conduced comprehensive NEMO lysine mutational analysis and identified the NEMO K270 residue as a crucial RANKL-regulation target. Specifically, NEMO harboring K270A mutation (NEMOK270A) elicits exacerbated osteoclastogenesis. More importantly, myeloid knock-in mice of the NEMOK270A that we generated displayed severe osteopenia and osteolysis. Mechanistically, autophagy is significantly decreased in NEMOK270A BMMs. Furthermore, proteomic screen identified interferon-stimulated gene-15 (ISG15) as a potential regulator of osteoclastogenesis and autophagy. Thus, our overarching hypothesis is: RANKL-induced binding of ISG15 to NEMO at K270 is essential to restrain osteoclastogenesis by assembling a negative-feedback response. We further posit that mutating K270 hinders this regulatory process leading to reduced autophagy and uncontrolled osteoclastogenesis. Our aims are: Aim 1: Determine the mechanism by which NEMO, through its K270 site, maintains physiologic and restrains pathologic/exacerbated osteoclastogenesis. Aim 2: Determine the role of RANKL-induced ISG15 as the ubiquitin-like protein that facilitates NEMO- K270-mediated autophagy and control of physiologic osteoclastogenesis.

抽象的：转录因子 NF-kB 在所有细胞类型中普遍表达，并且很容易被多种细胞激活。因子和细胞因子的基线 NF-kB 活性对于骨骼发育和生理细胞至关重要。相反，它在炎症过程中加剧且常常不受控制的活动会导致不良后果。具有主要功能障碍后果（包括骨质溶解）的有害影响，因此，针对 NF-kB 的治疗。不幸的是，大多数可用的治疗方法都受到高度重视。由于这种复杂且普遍存在的信号通路缺乏选择性，效率低下，因此至关重要 NF-kB 的有益功能与导致痛苦结果的有害作用一起被阻断。因此，解码 NF-kB 信号传导以识别分配的特定目标的需求尚未得到满足。信号特异性并区分生理和病理功能以解决这一关键问题。知识差距，我们专注于 RANKL 诱导的破骨细胞生成作为概念证明，并着手破译 NF-kB 分子机制并识别控制该机制的信号特异性分子特征破骨细胞祖细胞的反应并维持骨骼稳态 IKKγ/NEMO 作为一个平台，可在位点特异性地组装独特的信号激活或抑制该假设基于最新进展。暗示 NEMO 作为一个支架，整合信号分子以响应各种刺激赖氨酸 (K) 特定位点（参见图 2）。 SUMOylation，根据我们的新发现，ISGylation；附着泛素样蛋白的过程，我们对目标蛋白 ISG15（IFN 刺激基因）进行了全面的 NEMO 赖氨酸突变。分析并确定 NEMO K270 残基是关键的 RANKL 调节目标。携带 K270A 突变 (NEMOK270A) 会引起破骨细胞生成加剧，更重要的是，骨髓细胞生成加剧。我们生成的 NEMOK270A 敲入小鼠表现出严重的骨质减少和骨溶解。从机制上讲，NEMOK270A BMM 中的自噬显着降低。此外，蛋白质组筛选。确定干扰素刺激基因 15 (ISG15) 是破骨细胞生成和自噬的潜在调节因子。因此，我们的总体假设是：RANKL 诱导的 ISG15 与 NEMO 在 K270 处的结合对于我们进一步假设突变 K270 来抑制破骨细胞生成。阻碍这一调节过程，导致自噬减少和破骨细胞生成不受控制。目标 1：确定 NEMO 通过其 K270 位点维持生理功能的机制并抑制病理性/加剧的破骨细胞生成。目标 2：确定 RANKL 诱导的 ISG15 作为泛素样蛋白的作用，促进 NEMO- K270 介导的自噬和生理破骨细胞生成的控制。