Regulation of Osteoclastogenesis and Inflammatory Osteolysis

项目摘要/摘要 破骨细胞(OCs)是骨吸收细胞，起源于髓系OC祖细胞(OCPs)，是至关重要的 用于骨骼重塑和动态平衡。OC的分化和活性受到内在负离子的严格调控 反馈环(自动调节)和由其他细胞分泌的旁分泌因子，最明显的是骨保护素。 内在调节因子对于校准生理性肿瘤发生是至关重要的，其干扰会导致 不受控制的病理性成骨和骨溶解。在一项独立的研究中使用蛋白质组学研究，我们有 最近发现了干扰素刺激基因-15(ISG15)的一个新的自我调节作用，ISG15是一种泛素样小分子， 在《兽医创世纪》里。具体地说，我们发现RANKL诱导OCPs和OCS中ISG15的表达，并结合ISG15 致Nemo(也就是IKKg)下调核因子-kB信号。我们发现RANKL对OCPs的刺激 诱导OCPs分泌IFNA/b，参与并激活OCPs中的1型干扰素受体(IFNAR)信号 自身触发依赖于ISG15的局部自动调节负反馈回路，以限制 《肿瘤学》。这一机制似乎依赖于干扰素基因的刺激物(STING)，我们发现 是对RANKL的反应而表达的，对IFNA/b和ISG15的表达是必不可少的，并通过 数据显示，阻断STING或IFNA/b均可降低RANKL诱导的ISG15水平并加剧 《肿瘤学》。综上所述，这些观察表明，内源性ISG15通过其 与靶蛋白的经典结合，这是由高度特异的E1(Ube1L)、E2的顺序作用促进的 (UbcH8)，E3(Herc6)连接酶，一种被称为ISG的翻译后修饰，维持细胞 动态平衡。然而，ISG化是一种可逆的机制，在炎症条件下， ISG15由细胞大量产生，由分泌小泡分泌到细胞外(EC)空间，以及 通过与LFA1受体结合发挥细胞因子的作用。在这方面，我们表明，OCPs的炎症刺激与 细菌产物脂多糖抑制E1酶Ube1L的表达，并诱导大量ECISG15的分泌。 更令人惊讶的是，我们发现ISG15KO小鼠对内毒素的反应很差，骨溶解可以忽略不计 与脂多糖诱导的WT对应物的强壮性骨丢失相比。这一事件是依赖于肿瘤坏死因子的，这在Low身上很明显 WT小鼠ISG15KO血清中肿瘤坏死因子水平与血清中大量肿瘤坏死因子的比较。因此，ECISG15 似乎会加剧肿瘤的发生，并依赖于完整的内源性ISG15。 基于这些观察，我们的总体假设表明ISG15具有双峰函数： 生理条件下，ISG15与OC信号蛋白偶联以限制肿瘤的发生和维持 动态平衡，而炎症条件促进游离ECISG15的分泌，然后起到 炎性细胞因子可加剧肿瘤形成和骨溶解。为了验证这一假设，我们将：(1)阐明 细胞内ISG15抑制OCS的机制，以及(2)确定的机制(S) ECISG15加重基底骨肿瘤形成和炎性骨溶解

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