Regulation of Osteoclastogenesis and Inflammatory Osteolysis

破骨细胞生成和炎性骨质溶解的调节

• 批准号: 10681786
• 负责人: YOUSEF ABU-AMER
• 金额: $ 48.36万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10681786
• 关键词: Ablation; Acceleration; Autophagocytosis; Binding; Bone Resorption; Bone remodeling; Calibration; Cells; Data; Enzymes; Event; Extracellular Space; Family; Feedback; Genes; Goals; Homeostasis; IFNAR1 gene; In Vitro; Inflammatory; Injections; Interferon Type I; Interferon alpha; Interferon-beta; Interferons; Knockout Mice; Ligands; Ligase; Mediating; Metabolic; Modality; Molecular; Mus; Myelogenous; Myeloid Cells; NF-kappa B; Osteoclasts; Osteolysis; Osteopenia; Pathologic; Pathway interactions; Physiological; Post-Translational Protein Processing; Process; Production; Proteins; Proteomics; RANK protein; Regulation; Role; Secretory Vesicles; Serum; Signal Pathway; Signal Transduction; Signaling Protein; Site; Stimulator of Interferon Genes; TNF gene; Testing; Tumor necrosis factor receptor 11b; Ubiquitin; Virus; bone loss; cytokine; in vivo; member; neutralizing antibody; novel; osteoclast progenitor; osteoclastogenesis; overexpression; paracrine; progenitor; receptor; response; small molecule

Project Summary/Abstract Osteoclasts (OCs), the bone resorbing cells, arise from myeloid OC progenitors (OCPs) and are critical for bone remodeling and homeostasis. OC differentiation and activity are tightly regulated by intrinsic negative feedback loops (autoregulatory) and by paracrine factors secreted by other cells, most notably osteoprotegerin. Intrinsic regulators are critically important for calibrating physiologic OCgenesis, disruption of which leads to uncontrolled pathologic OCgenesis and osteolysis. Using proteomic studies in an independent study, we have recently identified a novel autoregulatory role of IFN stimulated gene-15 (ISG15), a ubiquitin-like small molecule, in OCgenesis. Specifically, we found that RANKL induces expression of ISG15 in OCPs and OCs, which binds to NEMO (a.k.a. IKKg) to down regulates NF-kB signaling. We found that stimulation of OCPs with RANKL induces IFNa/b secretion by OCPs, which engages and activates type 1 IFN receptor (IFNAR) signaling in OCPs themselves to trigger ISG15-dependent local autoregulatory negative feedback loop to limit the extent of OCgenesis. This mechanism appears to depend on STimulator of INterferon Genes (STING), which we find to be expressed in response to RANKL and is essential for IFNa/b, and ISG15 expression, and was validated by data showing that blocking either STING or IFNa/b diminishes RANKL-induced ISG15 levels and exacerbates OCgenesis. Collectively, these observations suggest that endogenous ISG15 inhibits OCgenesis through its classical binding to target proteins, which is facilitated by the sequential action of highly specific E1 (Ube1L), E2 (UbcH8), E3 (Herc6) ligases, a post-translational modification termed ISGylation that maintains cellular homeostasis. However, ISGylation is a reversible mechanism, whereby under inflammatory conditions, free ISG15 is generated in abundance by cells, secreted to the extracellular (EC) space by secretory vesicles, and acts as a cytokine by binding LFA1 receptor. In this regard, we show that inflammatory stimulation of OCPs with the bacterial product LPS inhibits expression of Ube1L, the E1 enzyme, and induces secretion of ample ECISG15. More surprisingly, we found that ISG15KO mice responded poorly to LPS and developed negligible osteolysis compared to robust bone loss by LPS-induced WT counterparts. This event was TNF-dependent, evident by low levels of TNFa in ISG15KO serum compared to copious amounts of TNF in serum of WT mice. Hence, ECISG15 appears to exacerbate OCgenesis and depends on intact endogenous ISG15. Based on these observations, our overarching hypothesis states that ISG15 has bi-modal functions: under physiologic conditions, ISG15 is conjugated to OC signaling proteins to limit OCgenesis and maintain homeostasis, whereas inflammatory conditions facilitate secretion of free ECISG15, which then acts as an inflammatory cytokine to exacerbate OCgenesis and osteolysis. To test this hypothesis, we will: (1) Elucidate the mechanism by which intracellular ISG15 inhibits OCs, and (2) Determine the mechanism(s) by which ECISG15 exacerbates basal OCgenesis and inflammatory osteolysis

项目总结/摘要 破骨细胞（OCs）是一种骨吸收细胞，起源于髓样破骨细胞祖细胞（OCPs）， 用于骨骼重塑和体内平衡OC的分化和活性受到内源性负调控， 反馈环（自动调节）和由其他细胞分泌的旁分泌因子，最显著的是骨保护素。 内源性调节因子对于校准生理性OCgenesis至关重要，其破坏导致 不受控制的病理性骨质疏松和骨质溶解。在一项独立的研究中，我们使用蛋白质组学研究， 最近鉴定了IFN刺激基因-15（ISG 15）的一种新的自身调节作用，ISG 15是一种泛素样小分子， 在OCgenesis。具体来说，我们发现RANKL诱导OCP和OC中ISG 15的表达，其结合 NEMO（又名NEMO）IKKg）下调NF-kB信号传导。我们发现RANKL刺激OCP 诱导OCP分泌IFN α/B，其参与并激活OCP中的1型IFN受体（IFNAR）信号传导 自身触发ISG 15依赖性局部自动调节负反馈回路，以限制 起源。这种机制似乎依赖于干扰素基因的STIMULATOR（STING），我们发现STING 在对RANKL的应答中表达，并且对于IFNa/B和ISG 15的表达是必需的，并且通过 数据显示阻断STING或IFNa/B降低RANKL诱导的ISG 15水平，并加剧 起源。总的来说，这些观察结果表明内源性ISG 15通过其自身的作用抑制OC发生。 与靶蛋白的经典结合，这是由高度特异性的E1（Ube 1 L）、E2 E3（Herc 6）连接酶，一种称为ISG化的翻译后修饰，其维持细胞内的 体内平衡然而，ISG化是可逆的机制，由此在炎症条件下，游离的ISG化是可逆的。 ISG 15由细胞大量产生，通过分泌囊泡分泌到细胞外（EC）空间， 通过结合LFA 1受体作为细胞因子发挥作用。在这方面，我们表明，OCP的炎症刺激， 细菌产物LPS抑制E1酶Ube 1 L的表达，并诱导样品ECISG 15的分泌。 更令人惊讶的是，我们发现ISG 15 KO小鼠对LPS的反应很差，发生的骨质溶解可以忽略不计 与LPS诱导的WT对应物的强骨丢失相比。该事件是TNF依赖性的，低 与WT小鼠血清中大量的TNF相比，ISG 15 KO血清中TNF α的水平。因此，ECISG 15 似乎加剧OC发生并依赖于完整的内源性ISG 15。 基于这些观察，我们的总体假设表明ISG 15具有双峰功能： 在生理条件下，ISG 15与OC信号传导蛋白缀合以限制OC发生并维持OC的表达。 体内平衡，而炎性条件促进游离ECISG 15的分泌，其然后充当免疫调节剂。 炎性细胞因子加剧骨质疏松和骨质溶解。为了验证这一假设，我们将：（1）阐明 细胞内ISG 15抑制OC的机制，以及（2）确定细胞内ISG 15抑制OC的机制。 ECISG 15加重基础骨形成和炎性骨溶解