Mechanisms of Physiologic and Pathologic Osteoclastogenesis

破骨细胞发生的生理和病理机制

• 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
• 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

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.

摘要： 转录因子nf-kB在所有类型的细胞中普遍表达，并很容易被许多 因子和细胞因子。基线核因子-kB活性对骨骼发育和生理细胞至关重要 功能。相反，它在炎症过程中加剧且经常失控的活动会导致不受欢迎的结果。 有害影响和严重的功能障碍后果，包括骨溶解。因此，针对核因子-kB的治疗 已经被广泛应用于抗击大多数炎症性疾病。不幸的是，大多数可用的治疗方法是 由于在这种复杂和无处不在的信号通路中缺乏选择性，导致效率低下，其中必不可少的 核因子-kB的有益功能在导致有害结果的有害影响的同时被阻断。 因此，有一种未得到满足的需求，即解码核因子-kB信号以识别指定的特定目标 信号的特异性，并区分生理和病理功能。要解决这一关键问题 知识缺口，我们将重点放在RANKL诱导的破骨细胞生成作为概念的证明，并着手 破译核因子-kB分子机制，并确定控制这一过程的信号特异性分子签名 破骨细胞祖细胞的反应和维持骨骼的动态平衡。我们假设IKK脚手架 IKKγ/NEMO作为一个特定部位组装独特信号激活或抑制的平台 以细胞和刺激的方式形成的蛋白质复合体。这一假说是基于最近的进展。 暗示尼莫是一种支架，它整合了信号分子，以响应广泛的刺激 赖氨酸(K)特异性位点(参见图2)。这些修饰包括，赖氨酸多泛素化， 苏莫化，根据我们的新发现，ISG化；连接泛素样蛋白的过程， ISG15(干扰素刺激基因)与靶蛋白结合。我们进行了全面的NEMO赖氨酸突变 分析并确定Nemo K270残基是关键的RANKL调控目标。具体地说，尼莫 携带K270A突变(NEMOK270A)可导致破骨细胞生成加剧。更重要的是，髓系 我们培育的NEMOK270A敲入小鼠表现出严重的骨量减少和骨溶解。 从机制上讲，NEMOK270A BMMS的自噬显著减少。此外，蛋白质组学筛选 发现干扰素刺激基因-15(ISG15)是破骨细胞生成和自噬的潜在调节因子。 因此，我们的主要假设是：RANKL诱导的ISG15与K270处的NEMO结合是必不可少的 通过组装负反馈反应来抑制破骨细胞的生成。我们进一步假设突变的K270 阻碍这一调控过程，导致自噬减少和不受控制的破骨细胞生成。我们的目标 目标1：确定尼莫通过其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。 目的2：确定RANKL诱导的ISG15作为泛素样蛋白促进NEMO-2的作用。 K270介导的自噬与生理性破骨细胞生成的调控。