Mechanisms of Physiologic and Pathologic Osteoclastogenesis

摘要： 转录因子NF-kB在所有细胞类型中普遍表达，并且容易被许多细胞因子激活。 因子和细胞因子。基线NF-kB活性对于骨骼发育和生理性细胞生长至关重要。 功能协调发展的相比之下，其在炎症期间加剧且通常不受控制的活性导致不期望的炎症。 有害影响，导致严重功能障碍，包括骨质溶解。因此，靶向NF-kB的疗法 已经被高度追求以对抗大多数炎性疾病。不幸的是，大多数可用的治疗方法是 由于在这种复杂和普遍存在的信号传导途径中缺乏选择性， NF-kB的有益功能被阻断沿着有害作用，导致有害结果。 因此，存在对解码NF-κ B信号传导以识别分配NF-κ B信号传导的特定靶标的未满足的需要。 信号特异性以及生理和病理功能之间区别。处理这个关键 由于知识上的差距，我们专注于RANKL诱导的破骨细胞生成作为概念验证，并着手 破译NF-kB分子机制，并识别控制这一机制的信号特异性分子特征。 反应破骨细胞祖细胞和维持骨骼稳态。我们假设IKK支架 IKKγ/NEMO是一个位点特异性组装独特信号激活或抑制的平台， 蛋白质复合物的细胞和刺激特异性的方式。这一假设是基于最近的进展 暗示NEMO作为一个支架，整合信号分子，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聚泛素化， SUMO化，根据我们的新发现，ISG化;一个连接泛素样蛋白的过程， ISG 15（IFN刺激基因）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残基为关键的RANKL调节靶标。具体而言，NEMO 携带K270 A突变（NEMOK 270 A）的小鼠可加重破骨细胞生成。更重要的是， 我们产生的NEMOK 270 A基因敲入小鼠表现出严重的骨质减少和骨质溶解。 从机制上讲，自噬在NEMOK 270 A中显著降低。此外，蛋白质组学筛选 确定干扰素刺激基因-15（ISG 15）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ISG 15与NEMO在K270处的结合对于 通过组装负反馈反应来抑制破骨细胞生成。我们进一步证实突变的K270 阻碍了这一调节过程，导致自噬减少和破骨细胞生成失控。我们的目标 目的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/恶化的破骨细胞生成。 目的2：确定RANKL诱导的ISG 15作为促进NEMO-1表达的泛素样蛋白的作用。 K270介导的自噬和生理性破骨细胞生成的控制。

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.