IgSF11 Signaling Controls Osteoclast Maturation and Pathogenic Bone Loss

IgSF11 信号传导控制破骨细胞成熟和致病性骨质流失

• 批准号: 10337682
• 负责人: YONGWON CHOI
• 金额: $ 35.75万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10337682
• 关键词: Affect; Biological; Biological Assay; Cell Surface Receptors; Cells; Colitis; Complex; Coupled; Data; Disease; Enzymes; Future; Genetic Transcription; Glycolysis; Homeostasis; Human; Immunoglobulins; Inflammation; Inflammatory; JAK2 gene; Link; Mass Spectrum Analysis; Mediating; Metabolic; Metabolism; Mind; Modeling; Modification; Molecular; Mus; Myelogenous; Nature; Osteoblasts; Osteoclasts; Osteogenesis; Pathogenicity; Pathway interactions; Patients; Phenotype; Phosphorylation; Phosphotransferases; Polyubiquitination; Process; Proteins; Pyruvate Kinase; Regulation; Role; SRC gene; Scaffolding Protein; Signal Pathway; Signal Transduction; TRAF6 gene; Testing; Therapeutic; Validation; base; bone; bone loss; bone resorbing activity; bone strength; clinically relevant; dextran sulfate sodium induced colitis; experimental study; inflammatory bone loss; inhibitor; member; mutant; novel; prevent; small molecule; src-Family Kinases

Under inflammatory conditions, bone destruction can be linked to excessive activity of bone-resorbing osteoclasts (OCs), which results not only from the differentiation of too many OCs, but also from over- maturation of OCs. While most current bone loss treatments prevent bone loss by reducing OC numbers, it may be better if future therapeutic strategies focus on targeting OC maturation rather than early OC differentiation to avoid inhibiting coupled bone formation that depends on interactions between bone-forming osteoblasts and OCs. In an effort to target maturation, we previously identified immunoglobulin superfamily member 11 (IgSF11) as a novel cell surface receptor that regulates OC differentiation but not new bone formation. To characterize IgSF11 signaling, we analyzed, by mass spectrometry, proteins phosphorylated after IgSF11 activation and identified Pyruvate kinase M2 (PKM2), the enzyme that catalyzes the last step of glycolysis, as a downstream target. This finding highlights a potentially greater than previously known determinative role for metabolic regulation during OC differentiation and inflammatory bone loss. We therefore propose the following specific aims: 1. Examine the role of IgSF11-PKM2 signaling in inflammatory bone loss. We will investigate OC-expressed IgSF11 in the context of inflammatory bone loss by using an LPS-induced model of bone loss. To test the contribution of PKM2-dependent effects, we will treat LPS-induced IgSF11-/- mice with small molecule modulators (TEPP-46, shikonin) of PKM2. Our preliminary data suggests that TEPP- 46 activation of PKM2 reduces DSS-induced bone loss. To examine whether IgSF11 expression affects colitis- associated bone loss, we will perform DSS-induced colitis experiments using IgSF11-deficient mice. We will also perform DSS-induced colitis experiments using IgSF11-/- mice treated with TEPP-46 or shikonin. These studies will be critical to establishing the intersection of IgSF11 and PKM2 contributions to clinically relevant inflammatory bone loss. 2. Characterization of IgSF11-PKM2 signaling mechanisms in osteoclast differentiation. We have formulated a four-step model of IgSF11-PKM2 function during OC differentiation, which we will test with the aid of hCD3-iFL, a retroviral (RV) construct to directly activate intracellular IgSF11 in differentiating OCs. We will first investigate possible crosstalk between RANK and IgSF11-PKM2, which we speculate is mediated by TRAF6-dependent K63-linked polyubiquitination of the IgSF11 scaffold protein PSD- 95. Second, we aim to identify kinases proximal to the IgSF11-PSD-95 complex that phosphorylate PKM2. Third, we will use RV mutants to confirm the importance of various PKM2 modifications, PKM2 allosteric confirmation, and PKM2 subcellular localization to OC differentiation. Finally, PKM2 is a well-characterized enzymatic regulator of glycolysis, so we will employ metabolic assays and inhibitors to confirm the significance of this aspect of PKM2 function to OC differentiation. These studies will be critically important to initial validation and characterization of a putative IgSF11-PKM2 pathway and its function during OC differentiation.

在炎症条件下，骨破坏可能与骨吸收的过度活动有关。 破骨细胞(OCs)，这不仅是由于过多的OCs分化造成的，也是由于过度的 业主立案法团的成熟。虽然目前大多数的骨丢失治疗是通过减少OC数量来防止骨丢失，但它 如果未来的治疗策略侧重于OC成熟而不是早期OC，可能会更好 避免抑制依赖于成骨之间相互作用的偶联骨形成的分化 成骨细胞和成骨细胞。为了以成熟为靶点，我们以前发现了免疫球蛋白超家族 成员11(IgSF11)是一种新的细胞表面受体，可调节OC的分化，而不是新骨 队形。为了表征igsf11信号，我们用质谱仪分析了磷酸化的蛋白质。 经IgSF11活化并鉴定为丙酮酸激酶M2(PKM2)的酶催化最后一步 糖酵解，作为下游靶点。这一发现突出了一个潜在的比之前已知的更大的 在骨质疏松症分化和炎症性骨丢失中代谢调节的决定作用。因此，我们 提出以下具体目标：1.研究IgSF11-PKM2信号在炎症性骨丢失中的作用。 我们将通过使用脂多糖诱导的骨丢失来研究炎症性骨丢失中OC表达的IgSF11 骨丢失模型。为了测试PKM2依赖效应的贡献，我们将治疗内毒素诱导的IgSF11-/- 小鼠使用PKM2的小分子调节剂(Tepp-46，紫草素)。我们的初步数据显示，Tepp- 46激活PKM2可减少DSS所致的骨丢失。为了检查IgSF11的表达是否会影响结肠炎- 对于相关的骨丢失，我们将使用IgSF11缺陷小鼠进行DSS诱导的结肠炎实验。我们会 使用Tepp-46或紫草素治疗的IgSF11-/-小鼠也进行DSS诱导的结肠炎实验。这些 研究对于确定IgSF11和PKM2对临床相关的贡献的交集是至关重要的 炎症性骨丢失。2.破骨细胞中IgSF11-PKM2信号传导机制的研究 差异化。我们建立了IgSF11-PKM2在OC分化过程中功能的四步模型， 我们将在hCD3-iFL的帮助下进行测试，hCD3-iFL是一种逆转录病毒(RV)结构，可以直接激活细胞内的IgSF11 区分OCS。我们将首先调查RANK和IGSF11-PKM2之间可能的串扰，我们 推测是由依赖TRAF6的K63连接的IgSF11支架蛋白PSD-的多泛素化所介导的。 95.第二，我们的目标是识别靠近IgSF11-PSD-95复合体的磷酸化PKM2的激酶。 第三，我们将使用RV突变体来确认各种PKM2修饰、PKM2变构的重要性 PKM2亚细胞定位对OC分化的影响。最后，PKM2是一个很好的特征 糖酵解的酶调节作用，因此我们将使用代谢检测和抑制剂来确认其意义 PKM2在OC分化中的这一方面作用。这些研究将对初始阶段至关重要 一个可能的IgSF11-PKM2通路的验证和特征及其在OC分化过程中的功能。