Mechanisms of Physiologic and Pathologic Osteoclastogenesis

破骨细胞发生的生理和病理机制

• 批准号: 10380048
• 负责人: YOUSEF ABU-AMER
• 金额: $ 33.21万
• 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 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10380048
• 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; Sumoylation Pathway; 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; osteoclast progenitor; osteoclastogenesis; 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.

抽象的： 转录因子NF-KB在所有细胞类型中均普遍表达，并且很容易被许多细胞激活 因素和细胞因子。基线NF-KB活性对于骨骼发育和生理细胞至关重要 功能。相比之下，炎症期间的加剧且经常不受控制的活动导致不希望的 有害作用带有主要功能失调的后果，包括骨溶解。因此，针对NF-KB的疗法 已被高度追求打击大多数炎症性疾病。不幸的是，大多数可用的疗法是 由于缺乏选择性，在这种复杂和普遍的信号传导途径中效率低下 NF-KB的有益功能与有害效应并导致有害结果的有害作用被阻止。 因此，没有必要解码NF-KB信号来识别分配的特定目标 信号特异性和生理和病理功能之间的区别。解决这个关键 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分子机械，并识别控制这一点的信号特异性分子特征 破骨细胞祖细胞的反应并保持骨骼稳态。我们假设IKK脚手架 IKKγ/NEMO充当一个平台，该平台特定地组装独特的信号激活或抑制 细胞和刺激特定方式中的蛋白质复合物。该假设基于最近的进步 将Nemo隐含为脚手架，该支架会响应于广泛的刺激范围内整合信号分子 赖氨酸（K）特定位点（请参阅图2）。这些修饰包括赖氨酸多泛素化， 根据我们的新发现，Isgylation；连接泛素样蛋白的过程， ISG15（IFN刺激的基因）靶向蛋白质。我们进行了全面的Nemo赖氨酸突变 分析并确定Nemo K270居住是至关重要的RANKL调节目标。具体来说，尼莫 携带K270a突变（NeMok270a）会加剧破骨细胞生成。更重要的是，髓样 我们产生的NeMok270a的敲入小鼠表现出严重的骨质减少和骨溶解。 从机械上讲，NEMOK270A BMM的自噬显着改善。此外，蛋白质组学筛选 鉴定出干扰素刺激的基因15（ISG15）是破骨细胞生成和自噬的潜在调节剂。 这是我们的总体假设是：在K270上，ISG15与Nemo的RANKL诱导的结合对 通过组装负反馈反应来抑制破骨细胞生成。我们进一步确认突变K270 阻碍了这种调节过程，导致自噬减少和不受控制的破骨细胞生成。我们的目标 是：目标1：确定Nemo通过其K270站点保持生理的机制 并限制病理学/恶化的破骨细胞生成。 AIM 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。